Galectin-3 as a therapeutic target in pulmonary hypertension: Molecular mechanisms, drug development directions, and emerging clinical applications

This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 1 Galectin-3 as a therapeutic target in pulmonary hypertension: molecular mechanisms, drug development directions and emerging clinical applications Antonín Sedlář1, *, Pavla Bojarová2, František Kolář3, Vladimír Křen2, Lucie Bačáková1 1 Laboratory of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-142 00 Prague 4, Czech Republic 2 Laboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-142 00 Prague 4, Czech Republic 3 Laboratory of Developmental Cardiology, Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, CZ 142 00 Prague 4, Czech Republic Abstract Pulmonary hypertension (PH) remains a devastating cardiovascular disorder with limited targeted treatment. Galectin-3 has emerged as a promising therapeutic target due to its central role in vascular remodeling and inflammation. Galectin-3 aggravates intimal hyperplasia by affecting endothelial cells through various mechanisms. It also contributes to medial hypertrophy and adventitial thickening by activating vascular smooth muscle cells and adventitial fibroblasts. In PH, right ventricular myocardium can be remodeled through galectin-3-mediated activation of cardiac fibroblasts and impaired cardiomyocyte contractility. Galectin-3 also modulates accompanying inflammatory reactions. This review explores the molecular mechanisms of galectin-3 action in PH and evaluates the potential of galectin-3 inhibition as a therapeutic strategy. The role of other galectins in PH are discussed as well. Targeting galectin-3 represents a novel avenue in PH management, but still faces challenges such as drug specificity and clinical efficacy. Future studies should focus on optimizing the structure and performance of galectin-3 inhibitors for clinical application. Graphical abstract Abbreviations αSMA, alpha-smooth muscle actin; ADMA, asymmetric dimethylarginine; Akt, protein kinase B; AMPK, 5'-adenosine monophosphate-activated protein kinase; Bax, Bcl-2 associated X, apoptosis regulator; Bcl-2, B-cell lymphoma 2; bFGF, basic fibroblast growth factor; CCL2, chemokine (C-C motif) ligand 2; CD, cluster of differentiation protein; CDK2, cyclin-dependent kinase 2; CHD, congenital heart disease; CRD, carbohydrate recognition domain; Creb, cyclic AMP response element binding protein; CRP, Creactive protein; CSE, cigarette smoke extract; CTD, connective tissue disease; CTEPH, chronic thromboembolic pulmonary hypertension; CTLA-4, cytotoxic T-lymphocyte antigen 4; CXCL1, This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 2 chemokine (C-X-C motif) ligand 1; DLL4, delta-like 4; ECM, extracellular matrix; ECs, endothelial cells; EMRP, modified rhubarb pectin; EndMT, endothelial-to-mesenchymal transition; eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; ERK1/2, extracellular signal-regulated kinases 1/2; FAK, focal adhesion kinase; FOXM1, forkhead box protein M1; G-CSF, granulocyte colonystimulating factor; GM, galactomannan; GM-CSF, granulocyte-macrophage colony-stimulating factor; GSK-3β, glycogen synthase kinase-3 beta; HPMECs, human pulmonary microendothelial cells; Hsp, heat shock protein; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular cell adhesion molecule-1; IgE, immunoglobulin E; IL, interleukin; IPAH, idiopatic pulmonary arterial hypertension; IPF, idiopatic pulmonary fibrosis; JAG1, Jagged1; JNK, c-Jun N-terminal kinase; LacNAc, NAcetyllactosamine; LAG-3, lymphocyte-activation gene 3; LC-3B, microtubule-associated proteins 1A/1B light chain 3B; LLPS, liquid-liquid phase separation; LV, left ventricle; MAPK, mitogen-activated protein kinase; MCAM, melanoma cell adhesion molecule; MCP, modified citrus pectin; MCP-1, monocyte chemoattractant protein-1; MCT, monocrotaline; MMP, matrix metalloproteinase; mPAP, mean pulmonary artery pressure; mRNA, messenger ribonucleic acid; mTOR, mammalian target of rapamycin; NADPH, nicotinamide adenine dinucleotide phosphate; NASH, non-alcoholic steatohepatosis; NF-κB, nuclear transcription factor kappa B; Notch, neurogenic locus notch homolog protein 1; Nox4, NADPH oxidase 4; NTS, N-terminal segment; oxLDL, oxidized low-density lipoprotein; PAAT, pulmonary artery acceleration time; PAECs, pulmonary artery endothelial cells; PAH, pulmonary arterial hypertension; PAFs, pulmonary adventitial fibroblasts; PASMCs, pulmonary artery smooth muscle cells; PAVTI, pulmonary arterial velocity time integral; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PH, pulmonary hypertension; PI3K, phosphoinositide-3-kinase; PKB, protein kinase B; PRAS40, proline-rich AKT1 substrate 1; RhoA, Ras homolog family member; RNA, ribonucleic acid; ROS, reactive oxygen species; RV, right ventricle; RVP, right ventricular systolic pressure; RVSP, right ventricular systolic pressure; Snail, Zinc finger protein SNAI1; Src, non-receptor tyrosin kinase; SRF, serum response factor; SSc, systemic sclerosis; TCR, T-cell receptor; TDG, thiodigalactosides; TGFβ1, transforming growth factor beta; Th, T helper cell; TNFα, tumor necrosis factor-alpha; TRPC1/4, transient receptor potential canonical 1/4; VCAM-1, vascular cell adhesion molecule-1; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; VEGFR1, VEGF receptor 1; VEGFR2, VEGF receptor 2; VSMCs, vascular smooth muscle cells; VTI, velocity time integral; WNK1, lysine deficient protein kinase 1; Wnt, Wingless/Int-1; WOS, web of science; YAP, yes-associated protein 1; ZEB1, Zinc finger E-box-binding homeobox 1; ZO-1, zonula occludens-1 Keywords: biomarker; carbohydrate-based inhibitor; drug development; fibrosis; galectin-3; pulmonary hypertension; vascular remodeling 1. Introduction Galectins are a conserved group of proteins typical of binding β-galactoside sugars. In terms of their classification in the system of biomolecules, they belong to lectins, i.e. carbohydrate-binding (glyco)proteins that specifically interact with carbohydrate epitopes in other molecules, such as those on the cell surface. Lectins are ubiquitous molecules in nature, being of plant, fungal, or animal origin. To date, 16 different galectin proteins have been identified in mammals. Galectins are also found in other organisms of the vertebrate subphylum, such as fish, birds, and amphibians, as well as in invertebrates and fungi [1,2]. Some of them exclusively occur in particular species. Twelve types of galectins have been found in humans so far. These are galectin-1, -2, -3, -4, -7, -8, -9, -10, -12, -13, -14, and -16, many of which are only expressed in certain tissues [3-6]. Galectin-1 and -3 are present in virtually all tissues of the body [7]. It is fair to say that galectin-3 is the most targeted and also the best-studied member of the galectin family, judging by the number of studies carried out on galectin-3. According to the WOS database, the number of publications on galectin-3 has been steadily increasing since the 1990s, and This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 3 over the last decade, the number of publications has regularly ranged between 400 and 550 per year, with a total of 8043 publications as of January 2025 (Figure 1). The second most studied galectin is galectin-1 with a publication count of 3085. The discovery of galectin-3 dates back to the early 1980s, when it was detected in mouse macrophages [8]. At the time of the discovery of galectin-3, its nomenclature was highly fragmented and this protein was presented under a variety of different names; e.g., Mac-2 [8], Carbohydrate binding protein 35 [9] or IgE-binding protein [10]. The names originated in the individual laboratories depending on the phenomenon studied and the experimental conditions, but then the name was standardized to galectin-3 [11]. The great interest in galectin-3 stems from its ubiquitous occurrence in the human body but is also due to its unique structural and biochemical properties. It is a globular protein consisting of an Nterminal domain and a C-terminal domain capable of binding sugars (carbohydrate-recognition domain; CRD). Galectin-3 is the only member of the wide group of galectin family with a unique chimeric structure. The other two groups of galectins are the tandem repeat group, comprising all galectins containing two distinct CRDs linked by a peptide linker (galectin-4, -6, -8, -9, and -12), and the prototypical single CRD galectin group comprising all remaining galectins [2]. The structure of galectin3 consists of a 21-amino-acid-long N-terminal segment (NTS) with two serine residues susceptible to phosphorylation, bound to nine non-triple-helical collagen-like Pro/Gly-rich repeats comprising sites susceptible to the cleavage by proteases. The structure is terminated by a CRD containing two tyrosine residues, optionally phosphorylated. The N-terminal domain, which comprises the NTS and most of the collagen-like repeats, serves for non-canonical secretion of the protein into the extracellular space [12]. The CRD consists of eleven β-folded sheets, six of which form a binding site for simple carbohydrates (the so-called S-face) and five opposing sheets form the so-called F-face, which can bind more complex sugars [13]. The F-face can transiently interact with the N-terminal domain [14]. In solution, galectin-3 occurs as a monomer, and at higher concentrations or in the presence of oligovalent ligands, it forms oligomers, such as pentamers, which can thus bind multiple ligands and form galectin-crosslinked protein network [15], which is a major mechanism behind its unique biological effects [16]. Some studies have observed the formation of dimers [17] or aggregation into larger structures [18]. A recent study has explained the oligomerization of galectin-3 on the cell membrane by a mechanism more reminiscent of liquid-liquid phase separation (LLPS), which is a process of separation of homogenous solution into two or more liquid phases [16]. LLPS is currently applied as the most accurate model for the formation of membrane-free cell organelles and intracellular compartments [19]. Galectin-3 is involved in a wide range of physiological and pathophysiological processes. To date, a large number of reviews have been published summarizing the role of galectin-3 in adhesion [20], cell growth, differentiation, apoptosis, cell cycle, and cell proliferation [21,22], as well as papers focusing on pathophysiology, i.e., the involvement of galectin-3 in diseases like cancer [23-25], a range of fibrotic diseases [26], inflammatory diseases [27] or cardiovascular diseases such as heart failure, atherosclerosis or arterial hypertension [24,28-30]. In recent years, the role of galectin-3 in pulmonary hypertension, a lethal cardiovascular disease affecting a significant proportion of the population, has been investigated, which will be thoroughly described and summarized in this review. Galectin-3 has been identified as a key player in vascular remodeling, fibrosis, and inflammation, making it a potential therapeutic target. This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 4 Figure 1: Graph showing the increase in the number of articles on galectin-3 per year since 1994. The articles were searched in all fields under the keyword "galectin-3". Publication Report graphic is derived from Clarivate Web of Science, Copyright Clarivate 2025. All rights reserved. 2. Pulmonary hypertension Pulmonary hypertension (PH) is a serious cardiovascular disease characterized by elevated mean pulmonary artery pressure measured by right heart catheterization, exceeding 20 mmHg at rest [31]. Its prevalence in the population is around 1%. This disease is particularly characteristic of the elderly population over 65 years of age, where the prevalence can be as high as 10%. The initial manifestations include non-specific symptoms such as dizziness, ankle swelling, chest pain, or palpitations. The prognosis of patients diagnosed with PH is discouraging since most of them die 3 years after diagnosis. In the later stages, the increased pressure causes remodeling of the right ventricular (RV) myocardium and pulmonary arteries, leading to heart failure and death. Pathophysiological changes can occur at all levels of artery branching, from the largest pulmonary arteries to the precapillary arterioles and capillaries, leading to occlusion and loss of peripheral volume of vessels. The changes are caused by varying degrees of endothelial cell damage and increased proliferation, muscular hypertrophy, and fibrosis. The changes may be accompanied by the formation of concentric and plexiform lesions, hypertrophy, and muscularization in the tunica intima. Hyperproliferation of vascular smooth muscle cells and the accumulation of extracellular matrix (ECM) proteins occurs, along with fibrosis and tissue thickening in the tunica adventitia and fibrotization of the RV myocardium. PH is a heterogeneous disease. Depending on the cause of the disease, patients with pulmonary hypertension can be divided into 5 groups. The first group is pulmonary arterial hypertension (PAH), which includes patients with pre-capillary PH. The second group of patients, in whom left-sided heart failure is the cause, has the highest incidence in the population. In the third group of patients, PH is caused by hypoxia, which is primarily triggered by lung diseases (sleep apnoea, chronic obstructive pulmonary disease, pulmonary fibrosis; it can also arise from staying at high altitudes). The fourth group includes patients with chronic thromboembolic pulmonary hypertension (CTEPH), which is associated with blood clotting diseases. In the fifth group of patients, the mechanism of PH is unclear and/or multifactorial; the development of the disease is caused, e.g., by cancer, kidney diseases, blood diseases, and chronic inflammation [32-36]. Galectin-3 is overexpressed in lung and heart tissue in human patients with PH of different etiology as well as in animal models of PH both on mRNA and protein levels [37-44]. Its elevated This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 5 expression is not only an accompanying phenomenon of pathophysiological processes in tissues during PH, but is directly functionally linked to the development and manifestations of the disease. Studies to date point to a potential role of galectin-3 as a pharmacological target in the treatment of PH, yet new knowledge is still needed on its precise molecular mechanisms of action in pathophysiological cardiovascular remodeling. 2.1. Galectin-3 and endothelial cell layer – role in intimal hyperplasia and thickening Endothelial cells (ECs) are among the main cellular players in PH-associated remodeling of vascular tissue. ECs form the lining of blood vessels and are the main component of the tunica intima. The main function of the endothelium is to form a barrier between the blood and the surrounding tissue, preventing blood clotting, leukocyte and platelet adhesion. Endothelial dysfunction in PH is usually caused by damage, whether mechanical or chemical, due to toxic substances, drugs, medications, or due to hypoxia. The dysfunctional endothelium is activated, an inflammatory reaction is generated in the subendothelial layer and a process of pathological intimal thickening (fibrosis) is triggered, which is a typical manifestation of atherosclerosis [45], and also a characteristic phenomenon of pulmonary hypertension [46]. In damaged and dysfunctional endothelium, there is an imbalance in the production of vasodilator and vasoconstrictor substances, causing chronic vasoconstriction, as well as increased secretion of growth factors stimulating cell proliferation and inflammatory mediators and production of adhesion molecules, e.g., E-selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule (ICAM-1), eventually leading to the recruitment of immune cells into vascular tissue and formation of inflammatory environment. Paracrine secretion of bioactive molecules also stimulates adjacent smooth muscle cells to proliferate, which also contributes to intimal thickening [47]. Endothelial dysfunction is involved in the pathophysiological remodeling of vascular tissue in almost all PH groups [48]. ECs express galectin-3. Other proteins of the galectin family abundantly expressed by ECs at both the RNA and protein levels comprise galectin-1, galectin-8, and galectin-9. In contrast, the genes for galectin-2, galectin-4, and galectin-12 are weakly transcribed in ECs. The expression of galectins can vary depending on the localization of the endothelium in tissues and organs; e.g., galectin-3 shows low expression in the endothelium of the liver and intestine [49]. Galectin-3 is present in ECs on the cell membrane surface and in the cytosol, particularly in perinuclear regions [50-52]. The migratory and angiogenic potential of ECs plays a crucial role in the development of PHrelated vascular remodeling not only in relation to the tunica intima in plexiform lesions formation [48,53], but also in the context of tunica adventitia during excessive angiogenesis and formation of a dense network of vasa vasorum surrounding the pulmonary arteries [54]. In ECs, both endogenous and exogenous galectin-3 have pro-angiogenic effects in vitro and also in vivo [55-59]. Angiogenesis can be activated by galectin-3 binding to αVβ3 integrin, which is a cell-matrix adhesion receptor. Binding to αVβ3 integrin activates the focal adhesion kinase (FAK) signaling pathway and pathways associated with basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which are critical for angiogenesis and cell migration [56]. Receptors regulating galectin-3-induced angiogenesis are also receptors of VEGF, namely VEGFR1 and VEGFR2, with the involvement of extracellular signal-regulated kinases 1/2 (ERK1/2) and heat shock protein 27 (HSP27) as downstream mediators of the pathway, but without activation of protein kinase B (Akt), nonreceptor tyrosine kinase (Src), and FAK [58]. Galectin-3 activates VEGFR1 and 2 receptors and, due to its oligomeric nature, forms cross-links with VEGFR1/2 receptors in the form of a galectin-glycan lattice. This regulates receptor trafficking and inhibits receptor internalization. In this way, the receptors remain on the cell surface at higher density and for a longer time, leading to increased receptor signaling [57,58]. The same mechanism, i.e., cross-linking and receptor clustering on the membrane, is probably involved in the binding of galectin-3 to integrin αVβ3 [56]. Angiogenesis may also be This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 6 stimulated by the interaction of galectin-3 with aminopeptidase N/CD13 [51]. VEGFR2-independent Notch signaling may also be stimulated. In this case, galectin-3 has a kind of dual role. On the one hand, it activates the delta-like 4 (DLL4) ligand, which regulates excessive endothelial sprouting, but on the other hand, it also activates the Jagged1 (JAG1) ligand, which in turn is pro-angiogenic [60]. Furthermore, galectin-3 also exerts chemotactic effects [55,61] and promotes the migration of ECs [62,63]. The pro-migratory effect is regulated by galectin-3 binding to the melanoma cell adhesion molecule (MCAM) receptor CD146, an immunoglobulin-like adhesion molecule that regulates cell proliferation and migration [62-64]. Galectin-3 also plays an important role in the adhesive properties of ECs, where it may be involved in cell-substrate adhesion [50], but also in cell-cell adhesion [65-67]. Interestingly, in addition to integrins α2β1 and α5β1, galectin-3-dependent adhesion to the substrate or extracellular matrix is mediated mainly by integrin αVβ3. After adhesion to galectin-3, the cells develop a specific morphology in vitro, which is stellate-shaped and has many protrusions, which could indicate their pro-migratory and pro-angiogenic phenotype [50]. It is also interesting that in the case of soluble exogenous galectin-3, the interaction with αVβ3 is due to the binding to the β-1,6-branched sugar groups on this glycoprotein receptor. In the case of galectin-3 adsorbed on the culture surface in vitro, the interaction with the integrin receptor appears to be independent of the carbohydratebinding capacity of galectin-3 [50,56]. Proliferation status and resistance or susceptibility to apoptosis in ECs is another important property that plays a key role in the development of PH. In the initial phase of PH, ECs are damaged and dysfunctional, which may be caused by a number of factors such as hypoxia, exposure to toxins, drugs and inflammatory mediators, or mechanical damage caused by increased blood pressure due to pathological features of arterial blood flow such as turbulence and pulsatile shear stress. ECs are first damaged and apoptotic processes are triggered, while in the later stages of the disease ECs in the tunica intima acquire resistance to apoptosis and show increased proliferation [68]. In human umbilical vein endothelial cells (HUVECs) and EA.hy926 endothelial cell line, exogenous galectin-3 stimulated proliferation [58]. The positive effect of galectin-3 on proliferation may also be influenced by its ability to activate the FAK signaling pathway [56], which is essential for cell survival and cell growth [69]. In pulmonary artery endothelial cells (PAECs), however, the effect of exogenous galectin on cell proliferation was slightly negative, which may be due to the use of a different type of ECs and the different concentrations used [42,43]. In these cells, galectin-3 caused cell arrest in G0/G1 phase and inhibited the expression of cyclin E and cyclin-dependent kinase 2 (CDK2), important mediators of cell cycle progression [43]. Galectin-3 also had a slightly negative effect on HUVEC proliferation and this effect was further enhanced when combined with oxidized low-density lipoproteins (oxLDL) [67,70]. It has been found that the hypoxic environment results in increased expression of galectin-3, for example, in cancer cells, where it has been shown to promote angiogenesis and cell survival, and to inhibit apoptosis [71]. Hypoxia is one of the factors triggering group 3 of PH. It also stimulates the expression of galectin-3 in PAECs, where the process is mediated by the nuclear transcription factor kappa B (NF-κB) [42,43,72]. In turn, in endothelial progenitor cells (EPCs), its expression can be induced by cigarette smoke extract (CSE; [52]). Galectin-3 inhibited apoptosis in PAECs. It caused an increase in the expression of the anti-apoptotic protein Bcl-2 and the pro-autophagy protein LC-3B, and a decrease in the expression of caspase-3, although its effect on proliferation was slightly negative. The genetic knockdown of galectin-3 in cells reduced Bcl-2 production [43,73]. In contrast, it was observed that exogenous galectin-3 suppressed the expression of the antiapoptotic proteins Bcl-2 and Alix [42]. This is consistent with another study with HUVECs where exogenous galectin-3 caused cell apoptosis via the β1-Ras homolog family member A-Jun N-terminal kinase (β1-RhoA-JNK) pathway [70]. At the same time, it suppressed autophagy by inhibiting the signaling pathway through transient receptor potential canonical 1/4 (TRPC1/4) calcium channels and activating the protein kinase B/glycogen synthase This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 7 kinase-3 beta/mammalian target of rapamycin (PKB/GSK-3β/mTOR) pathway [42]. Autophagy is a process that may serve as a rescue regulatory mechanism in ECs to protect against apoptosis [74]. The 5'-adenosine monophosphate-activated protein kinase/mTOR (AMPK/mTOR) autophagy pathway is also involved in the interaction of galectin-3 with EPCs [52]. This correlates with findings from other studies that also implicate the role of autophagy in PH, where its inhibition leads to an endothelial phenotype susceptible to apoptosis [68]. In contrast, in EPCs damaged by cigarette smoke extract, increased expression of endogenous galectin-3 is associated with autophagic processes and endothelial damage. By suppressing its expression, dysfunctional cells recovered their capillary formation and migratory ability, the activity of endothelial nitric oxide synthase (eNOS) was restored, while the production of reactive oxygen species (ROS) in the cells decreased [52]. Another pathophysiological mechanism, by which ECs contribute to tissue remodeling in pulmonary hypertension is endothelial-to-mesenchymal transition (EndMT). This is a rather complicated mechanism, in which ECs gradually lose their characteristic markers and transition to a mesenchymal phenotype. This process is thought to be triggered by damage to the endothelium and increased secretion of inflammatory mediators. The ECs are released from the inner lining of the vessel due to a decreased expression of tight and adherens junction proteins such as VE-cadherin or zonula occludens-1 (ZO-1), they gradually migrate inward (i.e., toward the subendothelial layer containing smooth muscle cells), lose expression of endothelial proteins, and adopt a myofibroblast/smooth muscle cell phenotype, characterized by expression of collagen, alpha-smooth muscle actin (αSMA), transforming growth factor beta 1 (TGFβ1), and proinflammatory cytokines. This process has also been identified in human PH-remodeled vessels and in an MCT animal model of PH. It is believed that EndMT is one of the causes of plexiform lesions in PH. Plexiform lesions are formations composed of glomerular-like protrusions in the tunica intima and are a sign of advanced PH. They are formed by ducts bordered by endothelial cells. Inside the lesions, a large number of αSMA-positive cells of various origins are present: smooth muscle cells, myofibroblasts, or fibroblasts, but also be ECs transdifferentiated by the EndMT process [75-78]. Galectin-3 is one of the factors that may contribute to EndMT activation. In PH, the role of galectin-3 in EndMT was demonstrated in an MCT rat model. Knockdown of galectin-3 resulted in the restoration of VE-cadherin expression in the pulmonary arteries of diseased rats, the presence of which is usually lost in transforming ECs in the EndMT process. At the same time, αSMA expression was reduced. Hypoxia or secretion of inflammatory molecules can trigger the EndMT process. In PAECs damaged by hypoxia or stimulated by tumor necrosis factor-alpha (TNFα), silencing of galectin-3 led to the same phenomena as in vivo, i.e., a decrease in αSMA and TGFβ1 expression and restoration of VE-cadherin expression, while the expression of transcription factors involved in EndMT (Zeb-1 and Snail) was negatively regulated [73]. Similarly, upon addition of exogenous galectin-3 to cultured PAECs, αSMA production was activated by the Jagged1/neurogenic locus notch homolog protein 1 (JAG1/Notch1) signaling pathway, which is dependent on the transcription factors SRF (serum-response factor) and myocardin [43]. The same trend was observed in human pulmonary microendothelial cells (HPMECs), where the Akt/β-catenin signaling pathway played a role in galectin-3-mediated EndMT [79]. Galectin-3-mediated EndMT was also observed in HUVECs, where it was regulated by the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway [80]. The dysfunctional and activated endothelium is also involved in the immune response and formation of an inflammatory environment in PH. It produces adhesion molecules that attract and recruit immune cells to the tissue, while producing a number of inflammatory mediators that contribute to inflammation [48,81]. Cytokine secretion in endothelial cells is initiated by galectin-3 binding to the N-glycan groups of the CD146, triggering the receptor dimerization and activation of the Akt-mediated signaling pathway. Other proteins involved in the downstream signaling pathway are heat shock protein 60 (HSP60), lysine deficient protein kinase 1 (WNK1), cell cycle inhibitor p27, β- This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 8 catenin, transcription factor c-jun, p70 ribosomal S6 kinase, cyclic AMP response element binding protein (Creb), tumor suppressor protein p53 and proline-rich AKT1 substrate 1 (PRAS40) [62]. In EPCs, cytokine secretory activity can also be triggered by the interaction of galectin-3 with integrin β1 via the RhoA-JNK and NF-κB signaling pathways [70]. Galectin-3 induces the secretion of pro-inflammatory cytokines, such as TNFα, interleukins (IL), namely IL-1, IL-6, IL-8, granulocyte-macrophage colonystimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), chemokines, such as CCL2 (also referred to as monocyte chemoattractant protein 1, MCP1) and CXCL1, or immunoglobulin adhesion molecules, such as ICAM-1 [62,67,70,72,73]. Galectin-3 also plays a role in the endothelial metabolism of ROS, which also contributes to the inflammation in the tissue remodeled by PH. For example, in microvascular endothelial cells from obese rats, galectin-3-activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and ROS production [82]. It also stimulated ROS production in EPCs damaged with cigarette smoke extract [52]. In oxLDL-damaged HUVECs, galectin-3 increased ROS production through the activation of NADPH oxidase and decreased the activity of antioxidant enzymes [67]. Activated endothelium mediates the recruitment of immune cells into the vascular tissue. Monocytes produce and secrete galectin-3 into the extracellular space, facilitating their adhesion to endothelial cells [66,67]. The facilitation of adhesion might be due to galectin-3 binding to CD106 (VCAM-1)/CD105(Endoglin), which are crucial for mediating adhesion to the endothelium [62]. In addition, adhesion of monocytes may also be mediated by galectin-3 of endothelial origin [72]. From all these studies, it can be concluded that galectin-3 has mainly an activating effect on the ECs in the tunica intima, stimulating their angiogenesis, migration and exerting chemotactic effects. It is also involved in inducing apoptosis and regulating EC proliferation. Also, through the EndMT process, it activates cell transdifferentiation towards a myofibroblast phenotype, characterized by the expression of ECM proteins and smooth muscle markers and migratory phenotype. Moreover, it triggers the secretion of pro-inflammatory cytokines and chemokines, ROS production and expression of immunoglobulin adhesion molecules in the endothelium, which shapes the inflammatory environment in the diseased vascular tissue (Figure 2). This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 9 Figure 2: Proposed mechanism of action of galectin-3 in endothelial cell layer of pulmonary arteries in PH. Galectin-3 induces cell migration and angiogenesis leading to neointimal thickening and plexiform lesion formation (A). Galectin-3 triggers endothelial-to-mesenchymal transition. Endothelial cells gradually loose expression of endothelial markers and transition to a mesenchymal phenotype, causing neointimal thickening and plexiform lesion formation (B). Galectin-3 stimulates apoptosis and cell cycle arrest leading to dysfunctional endothelium (C). Endothelial cells are stimulated by galectin-3 to secrete cytokines, chemokines (D) and to produce reactive oxygen species (E). Galectin-3 also promotes leukocyte adhesion to endothelium (F). These factors contribute to the formation of a proinflammatory environment in vascular tissue. αSMA, alpha-smooth muscle actin; Akt, protein kinase B; Alix, Programmed cell death 6-interacting protein; Bcl-2, B-cell lymphoma 2; CCL2, chemokine (C-C motif) ligand 2; CD, cluster of differentiation protein; CDK2, cyclin-dependent kinase 2; CXCL1, chemokine (C-X-C motif) ligand 1; EndMT, endothelial-to-mesenchymal transition; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM-1, intercellular cell adhesion molecule-1; IL, interleukin; JAG1, Jagged1; JNK, c-Jun N-terminal kinase; This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 16 pressure (RVP), Fulton index, and the formation of fewer partially muscularized microvessels compared with wild-type mice suffering from PH. Moreover, a higher increase in PA pressure in response to acute hypoxia was measured in galectin-1 knockout mice with PH, indicating reduced vasoreactivity in wild-type mice expressing galectin-1 [147]. The exact mechanism of galectin-1 involvement in PH is still unknown. However, findings from other studies indicate that galectin-1 is upregulated in activated endothelium and exhibits an angiogenic effect via VEGF signaling pathways [148]. Interestingly, its angiogenic effect acts synergistically with galectin-3 [58]. In the endothelium, it also has a stimulatory effect on other processes such as cell proliferation and migration [148]. In VSMCs, it enhances their adhesion to the ECM, thereby limiting cell motility, while having a slightly negative effect on PDGF-induced cell proliferation [149]. The effect of galectin-1 on vasoreactivity demonstrated in a mouse model of PH [147] is likely due to its reduction of L-type calcium current by binding to a specific splicing variant of the CaV1.2 channel, thereby regulating the channel expression in the membrane while also stimulating its degradation. This ultimately leads to lower vasoconstriction in VSMCs [150,151]. Galectin-1 is also known to negatively regulate immune responses. This is mainly due to its effect on T cells, where it selectively causes apoptosis of specific subpopulations such as Th1 and Th17 helper T cells, which have pro-inflammatory effects. This causes a redirection of the immune response towards a type influenced more by Th2 cells [152]. Thus, although the genetic inhibition of galectin-1 in the case of PH has led to an improvement in the functional characteristics of the disease [147], in other cardiovascular diseases, such as acute myocardial infarction, inhibition of galectin-1 caused a stronger inflammatory response and pathophysiological remodeling of cardiac tissue [153], suggesting that this galectin has a rather regulatory function in cardiovascular diseases. Remarkably, galectin-1 might be involved in the activation of tissue remodeling in the MCT model of PH through interaction with MCT metabolite monocrotaline pyrrole in PAECs [154,155]. Galectin-8, a tandem-repeat galectin, is expressed in the endothelium in a variety of tissues. Both exoand endogenous galectin-8 can stimulate angiogenesis and endothelial tube formation. It is also involved in the adhesion of ECs to the substrate or to other cells and has a pro-migratory effect. These properties are due to their interactions with integrin and immunoglobulin receptors on the surface of endothelial cells [156]. It also triggers the secretion of inflammatory cytokines in ECs [157]. It affects the fibrotic processes, although its role can be both antifibrotic [158] and profibrotic [159]. It is also involved in the immune response in a complex way, having a more activating effect on T cells and stimulating the secretion of pro-inflammatory cytokines, whereas it has a regulatory effect on Bcells [160]. In PH, galectin-8 is overexpressed in PAECs. Its overexpression was observed in both human patients and animal models of PH. Interestingly, its expression was also altered in pulmonary artery smooth muscle cells. Galectin-8 appears to trigger ROS production and apoptotic processes in dysfunctional PAECs, leading to pulmonary vascular remodeling. Mutant galectin-8-/- mice developed only mild symptoms of PH compared to wild-type mice [161]. Tandem-repeat galectin-9 is also likely to be involved in pulmonary artery remodeling in PH. In ECs, it affects angiogenesis and proliferation depending on its concentration and localization in the cell and the expressed isoform [162]. It also mediates the activation of lung fibroblasts through TGFβ/Smad signaling [163]. It is significantly involved in immune responses and is expressed by many immune cell types [164]. Its association with PH seems to be dependent on its posttranslational modifications - it is not sulphenylated at cysteine 74 under pathophysiological conditions in PH. Nonsulphenylated protein is activated and it can trigger mast cell degranulation and inflammatory processes leading to pulmonary arteriolar remodeling. Thus, galectin-9 appears to exacerbate and amplify the inflammatory response under pathophysiological conditions of PH [165]. This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 17 Other galectins may also be involved in PH, but their involvement is still speculative. For example, levels of circulating galectin-10 were significantly lower in patients with systemic sclerosis. Galectin-10 levels were negatively correlated with right ventricular systolic pressure as well as with inflammatory markers. A decrease in galectin-10 levels, which negatively regulates immune responses, seems to be associated with an increase in inflammatory markers in patients with systemic sclerosis. The negative correlation with RVSP is explained by the regulatory effect of galectin-10. Galectin-10 also has a regulatory effect on Treg lymphocytes that may be impaired due to its low levels in serum. Treg dysregulation is one of the phenomena specifically observed in PH that contributes to tissue remodeling. Decreased galectin-10 expression is likely to be related to the development of immune response and tissue remodeling in PH [166]. 3. Galectin-3 as a PH biomarker Biomarkers circulating in the blood, such as endothelin-I, asymmetric dimethylarginine (ADMA), brain natriuretic peptide, cardiac troponins, C-reactive protein (CRP), or cytokines [167,168], are usually associated with the underlying molecular mechanism of PH. Galectin-3 triggers fibrotic and inflammatory processes and its appearance in the blood is due to its increased secretion mainly by macrophages in inflamed tissue but also by other leukocytes or activated endothelial cells, smooth muscle cells, and fibroblasts. It appears that galectin-3 may serve as a biomarker for a number of fibrotic diseases such as liver fibrosis or systemic sclerosis [169]. It may also be a biomarker for cardiovascular diseases such as heart failure. Galectin-3 has been approved by the Food and Drug Administration (FDA) as a prognostic biomarker of left ventricular dysfunction and chronic heart failure [170]. Galectin-3 levels can predict all-cause mortality in the general population [171]. However, in PH, compared to left-sided heart failure, fibrosis, and hypertrophy are more likely to develop in the right ventricle. A correlation between galectin-3 levels and RV tissue remodeling was suggested in a study by Shah et al. where galectin-3 levels were associated with high RVSP in people affected by heart failure [172]. A similar correlation of galectin-3 levels with pulmonary artery pressure was found in studies observing patients with systemic sclerosis [173]; chronic obstructive pulmonary disease (COPD) [174] or heart failure [175,176]. Galectin-3 has been detected in studies involving smaller numbers of patients (on the order of tens) with PH of different etiologies, belonging to different World Health Organization (WHO) groups of PH. Compared to the healthy population, elevated levels have been found in PAH group 1 of different etiologies [37,44,91,113,177-179] and in PH related to heart failure in obese subjects [180]. However, in the case of CTEPH (group 4), there was no significant difference compared with healthy subjects [181]. Galectin-3 concentrations in the patients from these studies are summarized in Table 1. Apparently, it is not possible to distinguish the PH of different etiologies according to the levels of galectin-3 in circulating blood. Although galectin-3 concentrations are significantly elevated in the majority of PH patients, there was no difference between idiopathic PAH (IPAH) and PAH associated with connective tissue disease (CTD-PH), that both belong to group 1 of pre-capillary PH [177]. Similarly, there was no significant difference between galectin-3 levels in patients with IPAH and PAH associated with congenital heart disease (PAH-CHD; [44]) or IPAH and systemic sclerosis-associated PAH patients [113]. The same trend occurred when comparing patients with PH in different groups defined by WHO [182-184]. The only case showing significant differences in galectin-3 concentrations was a study comparing patients with SSc-PAH and IPAH [185]. Existing data suggest that galectin-3 may serve as a predictive marker of a patient's expected response to therapy. Li and coworkers aimed to identify blood biomarkers indicating the severity of pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD, group 1). The results of this study suggest that although galectin-3 concentrations do not directly correlate with This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 18 mPAP levels, and therefore with the severity of disease manifestation, its levels may indicate the treatability of the disease, in terms of the need for surgery and iloprost administration [186]. On the other hand, in PH patients also suffering from Down syndrome, galectin-3 concentrations were slightly higher, although not statistically significant, in resolved PH cases [179]. In CTEPH patients, there were no changes in galectin-3 levels 6 months after balloon pulmonary angioplasty [181] and in PH patients, the treatment with trimetazidine did not alter galectin-3 concentrations in blood [187]. In patients with heart failure, the severity and development of PH did not correlate with galectin-3 levels, as determined by RVSP measurement [188]. Other studies underline the prognostic significance of galectin-3. In patients with PAH, galectin-3 correlated positively with diastolic and systolic functions and right ventricular morphological parameters [189]. In children and adults with PAH-CHD, a positive correlation with pulmonary artery pressure has been reported, which, however, was only medium or weak, respectively [91,178]. In patients with PAH, there was a clearly detectable difference in galectin-3 levels for each functional group according to the severity of symptoms [177,185]. The higher the galectin-3 levels, the higher the mortality rate in the cohort of patients with PH of various etiologies was observed [183,184]. On the other hand, a recent multicenter study in a large cohort of PAH patients failed to find an association between serum galectin-3 and mortality from the disease. Nor did galectin-3 levels differ between patient groups according to the REVEAL registry risk score [190]. Unfortunately, it appears that the potential of galectin-3 as a diagnostic and prognostic marker in PH is still controversial, and needs to be validated by further studies. A possible solution lies in the inclusion of galectin-3 in a larger panel of biomarkers. Recently, Griffiths and coworkers were able to create a computational predictive model based on a set of circulating biomarkers including galectin-3, ST2 cardiac stress biomarker, insulin-like growth factor-binding proteins (IGFBP1, IGFBP2, IGFBP4), endostatin, and 3 demographic parameters, which was able to predict disease severity and mortality in PAH patients while outperforming currently used clinical scoring like European Society of Cardiology (ESC), European Respiratory Society (ERS) or REVEAL, which require the use of invasive and timeconsuming methods [191]. Table 2. Summary of the studies on galectin-3, measured as a circulating biomarker of PH in control healthy individuals and patients with various types of PH. Control group PH group Significance Reference PH type Galectin-3 concentration 7.615 ± 0.3884, n=14 PAH (IPAH + CHDPAH) IPAH, n=18 CHD-PAH, n=5 13.07 ± 1.01 13.06 ± 0.96 12.33 ± 1.76 p< 0.01 (all vs. control group) [44] 8.5 ± 0.9, n=8 IPAH, n=41 CTD-PAH, n=16 12.2 ± 0.6 14.1 ± 1.6 p<0.05 (IPAH vs. control group) p<0.05 (CTD-PAH vs. control group) [177] n.a. PAH, n=37 HFpEF-PH, n=39 22.33 (18.94, 27.30)a 28.94 (21.67, 39.85)a n.s., p<0.07 [183] 10.1 (9.0, 12.74)a, n=10 PAH (IPAH + CTDPAH), n=12+3 17.3 (13.2, 21.1)a p<0.006 (vs. control group) [189] This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 19 7.17 ± 1.72, n=14 PAH, n=26 14.68 ± 3.82 p<0.001 (vs. control group) [37] 1.038 ± 0.507, n=25 (children) VSD with PH, n=24 VSD w/o PH, n=26 2.889 ± 1.083 1.941 ± 0.707 vs. VSD w/o PH and control group) p<0.001 (vs. control group) [178] Ctrl, n=10 IPAH, n=38 SSC-PAH, n=43 n.a. (graph) PAH vs. ctrl p=0.0021 IPAH vs. ctrl p=0.0005 SSc-PAH vs. ctrl p=0.0079 [113] 13.6 (10.816.0)a, n=25 CTEPH, n=57 13.2 (10.4, 16.8)a n.s. [181] n.a. PAH, n=2017 CTD-PAH, n=623 IPAH, n=870 10.129 (6.92514.073a 10.444 (7.15115.057)a 10.335 (7.01014.270)a n.s. [190] n.a. PAH , n=70 IPAH, n=37 SSc PAH, n=17 20.4 ± 7.8 17.9 ± 7.4 24.0 ± 8.2 SSc PAH vs. IPAH, p=0.005 [185] n.a. IPAH, n=13 PAH CTD, n=17 PAH CHD, n=11 PAH other, n=12 PH group 3, n=14 CTEPH, n=21 PH group 5, n=10 n.a. (graph) n.s. [184] Ctrl, n=20 CHD-PAH, n=42 n.a. (graph) p<0.01 [91] Down syndrome w/o PH, 20.2 (13.125.2)a, n=29 Down syndrome+PH, n=49 15.5 (10.6-22.7)a n.s. [179] n.a. PAH, n=1623 10.88 (7.25-15.48)a n.a. [191] The concentration of Gal-3 is given either as mean ± standard deviation or as median (interquartile range)a, and in ng/mL. CHD-PAH, congenital heart disease-associated PAH; CTD-PAH, connective tissue disease-associated PAH; HFpEF-PH, heart failure with preserved ejection fraction-associated PH; IPAH – idiopathic PAH; n, number of subjects; n.a., not available; n.s., non-significant; PAH, pulmonary arterial hypertension; VSD, ventricular septal defect (in children); w/o, without. 4. Galectin-3 inhibition and its clinical perspectives in the treatment of PH Simple carbohydrate inhibitors Galectin-3 binds β-galactosides, which are carbohydrates containing a terminal β-galactoside linkage, via its CRD. The typical and simplest representative of such sugars is lactose, naturally found in breast This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 20 milk. However, galectin-3 biological activity is mainly modulated by more complex oligosaccharides, such as N-acetyllactosamine (LacNAc) terminated glycans and polyNAc-lactosaminoglycans in glycoproteins [192]. In the hypoxic rat model of PH, LacNAc was administered to rats from the day they were first exposed to chronic hypoxia. Administration of this inhibitor for 3 to 4 weeks resulted in a significant decrease in functional parameters of PH and pulmonary artery remodeling, such as mean pulmonary artery pressure (mPAP), right ventricular systolic pressure (RVSP), and Fulton index, which is an indicator of the degree of right ventricular hypertrophy, and inversely to the recovery of pulmonary artery acceleration time (PAAT) and pulmonary arterial velocity time integral (PAVTI) that are shortened in PH (Table 2) [43,44]. LacNAc was successfully used to treat a rat model of monocrotaline-induced PH [110] and a mice pulmonary arterial banding (PAB) model of chronic RV pressure overload as well (Table 2) [113]. Also, pharmacological inhibition of galectin-3 with LacNAc prevented left ventricular dysfunction and hypertensive nephropathy in REN2 rats prone to heart failure [193,194]. However, in general, low-molecular-weight hydrophilic sugars like LacNAc or lactose are unsuitable as drugs due to their rapid absorption and metabolic degradation in vivo. Pectins and galactomannans Pectins, particularly galactose-rich polysaccharides, have attracted attention for their potential therapeutic effects against fibrosis and pulmonary hypertension (PH). These compounds, including galactomannans (GMs) and modified citrus pectins (MCPs), act as galectin-3 antagonists and interfere with processes that lead to tissue scarring and vascular remodeling. GMs such as GM-CT-01 (Davanat®), have been shown to inhibit galectin-3 and thereby attenuate fibrotic processes. Davanat® was shown to increase the activity of human tumor-infiltrating lymphocytes and to disrupt galectin-glycoprotein lattices, although the exact mechanisms are still under investigation [195]. In mouse models of non-alcoholic steatohepatitis (NASH), GM-CT-01 and another GM, GR-MD-02, reduced hepatocellular damage, inflammation, and fibrosis, with GR-MD-02 showing greater efficacy. Modified citrus pectins MCPs derived from citrus fruits have been studied for their antifibrotic properties. Various MCP formulations such as GCS-100 and PectaSol-C® have demonstrated the ability to inhibit galectin-3-mediated hemagglutination, reduce cardiac inflammation and attenuate organ fibrosis in animal models [196]. In a recent study, modified rhubarb pectin (EMRP) was identified as a potent galectin-3 inhibitor. In a mouse model of angiotensin II-induced myocardial fibrosis, treatment with EMRP resulted in a 57% reduction in left ventricular fibrosis, exceeding the 30% reduction observed with conventional MCP. This anti-fibrotic effect was associated with a reduced inflammatory response of the heart and preservation of cardiac function [197]. The role of pectins in pulmonary fibrosis has been further investigated through innovative delivery methods. Recent research has investigated the use of citrus pectin-coated poly(lactic-coglycolic acid) (PLGA) nanoparticles as an inhalable therapeutic agent. This approach significantly improved pulmonary fibrosis in vivo over a 24–72 hour period, suggesting that pectin-based therapies may be effective in targeting fibrotic lung tissue. The study also suggested that combining this delivery system with encapsulated antifibrotic drugs could improve therapeutic efficacy and patient adherence by reducing off-target effects and providing additional benefits [198]. However, clinical trials targeting galectin-3 with MCPs have produced mixed results. In a randomized placebo-controlled trial of participants with elevated galectin-3 levels and hypertension, treatment with MCPs had no significant effect on markers of collagen metabolism, echocardiographic measurements, or vascular function [199]. The antifibrotic effect of pectins is primarily attributed to their inhibition of galectin-3. Recent studies suggest that MCPs and GMs can bind to a non-canonical site of the carbohydrate recognition domain of galectin-3, and thus represent a new target for therapeutic intervention. However, discrepancies exist regarding the ability of pectins to inhibit galectin-3-mediated hemagglutination. This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 21 Further research is needed to clarify the exact mechanisms of action. Pectins, particularly GMs and MCPs, especially combined with innovative delivery methods, thus exhibit potential as antifibrotic agents by targeting galectin-3, reducing fibrosis and inflammation in various organ systems, including the heart and lungs. Nonetheless, mixed outcomes of clinical trials underscore the necessity for continued research to confirm their effectiveness and safety in human populations. The beneficial role of galactomannans and pectins has been proven also in the treatment of pulmonary hypertension (Table 2). In the reports by Barman et al., it has been shown that administration of GM-CT-01 or GR-MD-02 inhibitors for 4 weeks, starting with MCT administration, attenuated the severity of the developed PH. Functional and hemodynamic parameters, such as RVSP, Fulton index, RV thickness, and velocity time integral (VTI), reached values similar to those of control healthy animals. Interestingly, the decrease in RVSP and Fulton index occurred even when inhibitors were applied after the development of the disease (i.e., 3 weeks after monocrotaline administration), suggesting a possible application in patients with the already developed disease [39,40]. Similar treatment outcomes in the MCT model were achieved when modified citrus pectin was applied [41]. Thiodigalactosides Thiodigalactoside (TDG) derivatives are synthetic disaccharide inhibitors of galectin-3. In particular, compounds such as TD-139 (also known as GB0139, Galecto Inc. https://ir.galecto.com/) have been designed to target non-canonical sites within the CRD of Gal-3, specifically subsites B and E, potentially leading to allosteric modulation of galectin-3 activity [200]. Preclinical studies have shown the antifibrotic potential of TDG derivatives. In particular, TD-139 has been shown efficiency in reducing pulmonary fibrosis in mouse models. In one study, administration of TD-139 attenuated bleomycininduced pulmonary fibrosis, suggesting its potential as a therapeutic agent for fibrotic lung disease [201]. TD-139 (GB0139) has even been enrolled in clinical trials for the treatment of idiopathic pulmonary fibrosis (IPF). However, despite promising preclinical data, clinical trials were recently reported to have been halted as the drug failed to show sufficient efficacy in patients. This highlights the challenges of translating preclinical success into clinical efficacy and underscores the need for further research to optimize the therapeutic potential of TDG derivatives [202]. TDG-based inhibitors have been further tuned by systematic structural modifications, such as the introduction of fluorine substituents, bringing enhanced affinity and selectivity for galectin-3, which opens further pathways to high-efficacy inhibitors of galectin-3 useful for clinical treatment of fibrotic diseases [203]. However, ongoing research aims to clarify the therapeutic benefit of galectin3 inhibition in PH patients. Heparin-based inhibitors Heparin-based inhibitors, particularly chemically modified non-anticoagulant derivatives, have emerged as promising agents targeting galectin-3. Through specific modifications, these derivatives aim to maintain therapeutic efficacy while minimizing the anticoagulant effect. Modified heparin derivatives exhibited a high selectivity for galectin-3 without inhibiting other galectins such as galectin1, -4, and -8. In addition, they have no significant anticoagulant effect, which reduces the risk of bleeding complications associated with conventional heparin therapies [204]. In vivo studies using nude mouse models showed that these inhibitors significantly reduce galectin-3-mediated lung metastasis of human melanoma and colon cancer cells [204]. Although these results are promising, direct evaluation of these inhibitors in fibrotic disease models, particularly with respect to pulmonary fibrosis, remains an area for future research. While there are few direct studies of heparin-based galectin-3 inhibitors in PH models, the established role of galectin-3 in PH suggests that these inhibitors may offer therapeutic benefits in the treatment of the disease. This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 22 Neo-glycoconjugates Neo-glycoconjugates, particularly glycopolymers, are another class of promising galectin-3 inhibitors. Synthetic neo-glycoconjugates are designed to present multiple carbohydrate ligands, which further increases their binding affinity for galectin-3 [205]. A recent study by us [38] presented a series of glycopolymers derived from lactose and its glycomimetics, designed to inhibit galectin-3. These glycopolymers showed subnanomolar binding affinities to galectin-3 as determined by biolayer interferometry. In cellular models using cardiac fibroblasts and pulmonary artery smooth muscle cells, they significantly reduced the expression of markers of tissue remodeling associated with PH. In addition, these glycopolymers were internalized cellularly, indicating possible intracellular mechanisms of action. A previous study by Michel et al. [206] investigated lactose-functionalized dendrimers as galectin-3 inhibitors. Their results indicated that smaller dendrimers can inhibit cancer cell aggregation through competitive binding while larger dendrimers enhance aggregation by providing multiple galectin-3 binding sites. This dual behavior highlights the importance of molecular architecture in the development of effective galectin-3 inhibitors. The study by Sedlář et al. [38] extends these findings by focusing on glycopolymers with a high galectin-3 affinity and examining their effects in the context of PH. The observed downregulation of markers of tissue remodeling suggests that these glycopolymers may interfere with galectin-3-mediated signaling pathways involved in fibrosis and vascular remodeling. As the next step, in vivo studies are needed to confirm the therapeutic potential of these compounds and their safety in the clinical setting. Peptides Peptides can be easily prepared through recombinant expression or by automated synthesis. Depending on their structure and size, however, they may show immunogenicity, which is a potential drawback of this class of inhibitors. The NH₂-terminally truncated form of galectin-3, known as Gal-3C, has been studied in the context of galectin-3-related tumors and has shown potential as a therapeutic agent with a favorable safety profile. Studies have shown that Gal-3C can inhibit tumor growth and metastasis in orthotopic nude mouse models of human breast cancer [207]. In addition, research suggests that Gal-3C enhances the anti-cancer effects of bortezomib in mouse models of human multiple myeloma [208]. The efficacy of Gal-3C in the treatment of fibrosis and PH is unknown yet. Another peptide-based approach focuses on the galectin-3-binding peptide G3-C12. This peptide exhibits a high selectivity for galectin-3, making it a valuable tool for targeted therapy. Sun and colleagues investigated the potential of G3-C12 to inhibit intracellular galectin-3 in cancer cells and showed its efficacy in a large panel of galectin-3-associated effects [209]. By conjugating G3-C12 with a suitable drug carrier, such as a synthetic polymer, a targeted delivery system may be prepared that shows promise for in vivo application involving galectin-3, including organ fibrosis and heart failure. Genetic silencing The crucial role of galectin-3 in PH was also confirmed in animal models where its expression was genetically silenced. A functional role of galectin-3 has been demonstrated in galectin-3-/- mutant mice exposed to hypoxia [72] or after undergoing pulmonary arterial banding [113]. Genetic knockout of galectin-3 led to significant improvement of functional parameters in the rat MCT and SU5416/Hypoxia (Su/H) models [39,40]. Similarly, the use of lentiviral technology to genetically knock down the expression of galectin-3 in the rat MCT model of PH [73] and in a mouse shunt-associated PAH model [91] led to milder severity of the disease (Table 2). Table 2: Studies demonstrating the beneficial effect of galectin-3 inhibition in animal models of pulmonary hypertension. This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 23 PH Model/Animal Experimental conditions Mode of Gal-3 inhibition Outcome Reference Hypoxia/ Male Sprague-Dawley rats 10% O2 for 4 weeks LacNAc (5 mg/kg per day, intraperitoneally, 3 times a week for 4 weeks, during exposure to hypoxia) Decreased mPAP, RVSP, Fulton index and arterial hypertrophy [44] Hypoxia/ Male Wistar rats 10% O2 for 3 weeks LacNAc (5 mg/kg, intraperitoneally, 3 times a week, during exposure to hypoxia) Decreased pulmonary artery pressure, increased PAAT and PAVTI [43] MCT/ Male Sprague-Dawley rats MCT intraperitoneal injection, (60mg/kg) LacNAc (5 mg/kg every other day, starting the day after MCT injection) Decreased mPAP, RVSP and Fulton index, unaffected systemic blood pressure [110] PAB (chronic RV pressure overload model)/C56BL/6J mice and Gal3-/- mice Occlusion of the main pulmonary artery by titanium clip LacNAc (5 mg/kg/d, intraperitoneally, 3 times a week for 3 weeks, starting 7 days after the surgery) or Galectin-3 null mutant mice No change in RVFW, RVSP, RVEDP, CO, TAPSE and echocardiographic parameters. Decreased fibrosis in RV. [113] MCT/ Male Sprague-Dawley rats MCT intraperitoneal injection, (60mg/kg) GM-CT-01 or GR-MD-02 (60 mg/kg iv, twice weekly for 4 weeks starting at the time of MCT injection. Reversal protocol – 3 weeks after MCT treatment – twice weekly for another 3 weeks. Decreased blood vessel wall area, RVSP, Fulton index, RV thickness, increased VTI. Reversal protocol – decreased RVSP and Fulton index. [39,40] MCT/ Male Sprague-Dawley rats MCT intraperitoneal injection, 60mg/kg MCP (100 mg/kg once a day for 28 days in the drinking water, starting the day of MCT injection) Increased PAAT, decreased mPAP, right ventricular hypertrophy index, vessel wall thickness and area in small and middle size blood vessels [41] Hypoxia/ Male C57BL/6J mice and Gal3-/- mice 10% O2 for 4 weeks Galectin-3 null mutant mice Decreased RVSP and Fulton index [72] MCT or Su/H/ Male SpragueDawley rats MCT intraperitoneal injection, (60mg/kg) or injection of the VEGF receptor antagonist SU5416 (20mg/kg, sc.) followed by 3 weeks of hypoxia Gal-3 KO rats using CRISPR technology decreased RVSP, Fulton index, relative vessel wall area, RV thickness, increased VTI and CO [39,40] This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 24 CO, cardiac output; CRISPR, clustered regularly interspaced short palindromic repeats; Gal-3, galectin3; KD, knockdown; KO, knockout; MCP, modified citrus pectin; MCT, monocrotaline; mPAP, mean pulmonary artery pressure; N-Lac, N-acetyllactosamine; PAAT, pulmonary artery acceleration time; PAB, pulmonary artery banding; PAVTI, pulmonary arterial velocity time integral; RV, right ventricle; RVEDP, RV end distolic pressure; RVHI, right ventricular hypertrophy index; RVSP, RV systolic pressure; RVWF, RV free wall thickness; sc., subcutaneously; sh-Gal-3, short hairpin RNA silencing galectin-3 expression; sPAP, systolic pulmonary artery pressure; Su/H, SU5416/Hypoxia; TAPSE, tricuspid annular plane systolic excursion; TU, transduction units; VEGF, vascular endothelial growth factor; VTI, velocity time integral. 5. Conclusion and further perspectives Despite major advances in cardiovascular medicine, pulmonary hypertension is still a very serious and incurable disease. Treatment of pulmonary hypertension focuses only on attenuating the symptoms of the disease and prolonging life. Vasodilator drugs based on soluble guanylate cyclase (sGC) stimulators, endothelin receptor antagonists, phosphodiesterase 5 inhibitors nad prostacyclin analogues are given to patients to lower blood pressure [210]. They are mainly used to treat PAH and may, on the contrary, be inappropriate for the treatment of PH of other etiologies. Therapies for PH of other etiologies specifically target diseases that may cause PH, such as left-sided heart failure, COPD, sleep apnea or chronic thrombo-embolic pulmonary disease [211]. Galectin-3 acts as an important profibrotic factor in a number of cardiovascular diseases [29]. In the development of pulmonary hypertension, galectin-3 contributes to pathological remodeling of pulmonary arteries at all levels of branching. It also contributes to changes in right ventricular myocardium and influences the immune response accompanying PH. Galectin-3 plays a key role in the activation of fibrotic pathways. It induces endothelial-to-mesenchymal transition. During this process, endothelial cells acquire a mesenchymal phenotype characterized by increased TGF secretion [73]. In smooth muscle cells, it activates their switch to a transient phenotype characterized by increased proliferation, ECM protein production and TGF secretion [97]. Similarly, fibroblasts in the tunica adventitia and right ventricle are activated [37,109]. Galectin-3 stimulates TGF signaling, which is important for the activation of these processes [102]. Inhibition of TGF signaling has been shown to be beneficial in reversing the negative manifestations of pulmonary hypertension in animal models of PH [212,213]. In this regard, inhibition of galectin-3 appears to be another option for attenuating TGF signaling. Remodeling of arteries and RV in PH is also due to activation of immune cells and the establishment of an inflammatory environment in tissues [126]. Galectin-3 stimulates the production (10% O2) and 3 weeks of normoxia (21% O2) MCT/ Male Sprague-Dawley rats MCT injection into abdominal subcutaneous tissue (60 mg/kg), 3 weeks incubation Gal-3 KD rats - lentiviral vector injection (intratracheally, 50 µl, 2×108 TU/mL, 1 week before MCT treatment) lower RVSP, Fulton index, medial wall thickness and vascular muscularization [73] Shunt-associated PAH/ BALB/C mice Arteriovenous shunt between abdominal aorta and inferior vena cava made by surgery Tail vein injection of the lentiviral vector containing sh-Gal-3 (100 µl, 5×107 TU/mL, day 0 after the surgery) Decreased sPAP, RVHI, medial thickness [91] This preprint is made available under CC BY 4.0 international license at https://doi.org/10.5281/zenodo.17573995 25 of inflammatory mediators in pulmonary artery and right ventricle. From this perspective, inhibition of galectin-3 may be part of an anti-inflammatory therapy that appears to be effective in the treatment of some etiologies of PH [214]. Biological activity of galectin-3 can be blocked by different types of inhibitors. Some of them, such as thiodigalactoside TD-139 (GB0139) or MCPs, have entered clinical testing phases, but none of them has been approved for clinical use [199,202]. Recent results have shown that in patients with pulmonary fibrosis, TD139 has not shown sufficient efficacy to halt disease progression, indicating a still difficult and long way to go in the final clinical application of these inhibitors [202]. A promising strategy to overcome the ambiguous results from clinical trials might be the synthesis of neoglycoconjugates with specific molecular architecture [38,206]. Studies to date show that inhibition of galectin-3 targets a variety of molecular pathophysiological mechanisms that cause PH of different etiologies, making this protein a promising molecular target for pharmacological treatment [39,40,43,44]. Recent data also show that other galectins are involved in the pathophysiology of PH, namely galectin-1, galectin-8, and galectin-9 [147,161,165]. This fact may be exploited in the application of carbohydrate-based inhibitors, which are rather nonspecific for binding to individual galectin types, and a potential multitarget approach in the treatment of PH. Contributor roles: Conceptualization - A.S., L.B.; Data curation – A.S.; Funding acquisition – P.B., L.B.; Project administration – P.B., L.B.; Supervision – L.B.; Visualization – A.S.; Writing – original draft – A.S., P.B., F.K., V.K., L.B.; Writing – review & editing - A.S., P.B., F.K., V.K., L.B. Conflict of interest: Authors declare no conflict of interest. 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