Endothelial cell regulation of systemic haemodynamics and metabolism acts through the HIF transcription factors

Endothelial cell regulation ofsystemic haemodynamics andmetabolism acts throughtheHIF transcription factors Simon Lambden1, Andrew S. Cowburn2, David Macias1, Tessa A. C. Garrud3, Bernardo J. Krause4, Dino A. Giussani3, Charlotte Summers1† and Randall S. Johnson3,5*† Abstract Background: The vascular endothelium has important endocrine and paracrine roles, particularly in the regulation of vascular tone and immune function, and it has been implicated in the pathophysiology of a range of cardiovascular and inflammatory conditions. This study uses a series of transgenic murine models to explore for the first time the role of the hypoxia-inducible factors, HIF-1α and HIF-2α in the pulmonary and systemic circulations as potential regulators of systemic vascular function in normoxic or hypoxic conditions and in response to inflammatory stress. We developed a series of transgenic mouse models, the HIF-1α Tie2Cre, deficient in HIF1-α in the systemic and pulmonary vascular endothelium and the L1Cre, a pulmonary endothelium specific knockout of HIF-1α or HIF-2α. In vivo, arterial blood pressure and metabolic activity were monitored continuously in normal atmospheric conditions and following an acute stimulus with hypoxia (10%) or lipopolysaccharide (LPS). Ex vivo, femoral artery reactivity was assessed using wire myography. Results: Under normoxia, the HIF-1α Tie2Cre mouse had increased systolic and diastolic arterial pressure compared to litter mate controls over the day–night cycle under normal environmental conditions. VO2 and VCO2 were also increased. Femoral arteries displayed impaired endothelial relaxation in response to acetylcholine mediated by a reduction in the nitric oxide dependent portion of the response. HIF-1α L1Cre mice displayed a similar pattern of increased systemic blood pressure, metabolic rate and impaired vascular relaxation without features of pulmonary hypertension, polycythaemia or renal dysfunction under normal conditions. In response to acute hypoxia, deficiency of HIF-1α was associated with faster resolution of hypoxia-induced haemodynamic and metabolic compromise. In addition, systemic haemodynamics were less compromised by LPS treatment. Conclusions: These data show that deficiency of HIF-1α in the systemic or pulmonary endothelium is associated with increased systemic blood pressure and metabolic rate, a pattern that persists in both normoxic conditions and in response to acute stress with potential implications for our understanding of the pathophysiology of vascular dysfunction in acute and chronic disease. Open Access © The Author(s), 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. RESEARCH ARTICLES Lambdenetal. ICMx (2021) 9:28 https://doi.org/10.1186/s40635-021-00390-y Intensive Care Medicine Experimental *Correspondence: [email protected] †Charlotte Summers and Randall S. Johnson are joint senior authors 3 Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK Full list of author information is available at the end of the article Page 2 of 14 Lambdenetal. ICMx (2021) 9:28 Keywords: HIF-1α, HIF-2α, Blood pressure, Haemodynamics, Metabolism, Vascular endothelium Introduction The vascular endothelial cell has a central role in the cardiovascular system, and as such participates in the pathophysiology of multiple cardiovascular diseases [1, 2]. The endothelium contributes extensively to the regulation of vascular tone, permeability and blood flow through both direct synthesis of mediators such as nitric oxide and indirectly by regulating circulating vasoactive substances such as catecholamines and angiotensin II [3]. Dysregulation of these pathways has been associated with the development of both acute and chronic vascular dysfunction [4] and in critical illness, impaired endothelial function has been widely reported and associated with poor outcomes [5–9]. However, to date, mechanistic understanding of what drives the development of vascular endothelial dysfunction and the consequences of this are lacking. The hypoxia-inducible factor isoforms (HIF-1α and HIF-2α) control transcriptional activity of a significant number of genes. In hypoxic conditions, stabilisation and reduced turnover of HIF-1α and HIF-2α through inhibition of the oxygen-dependent prolyl hydroxylase isoforms results in heterodimer formation, leading to HIF-1β subunit and promoter binding to induce transcription. However, there is evidence that HIF stabilisation can be driven by other stimuli, including inflammation [10, 11] and, in vascular endothelial cells, shear stress [12]. These alternate pathways of HIF regulation may, for example, act in the progression of atherosclerosis [12] thus suggesting that a functional role for the HIF isoforms in both homeostatic and stress-induced conditions other than hypoxia is possible. HIF function in peripheral tissues is also involved in regulating systemic haemodynamics. Animals deficient in keratinocyte HIF isoforms display mild hypertension or hypotension, dependent on which isoform is deleted [13]. In animal models testing the impact of therapeutic stabilisation of HIF isoforms as a treatment for anaemia, a significant dose dependent reduction in blood pressure was observed following administration of such compounds [14]. To date, the role of the HIF isoforms in the vascular endothelium itself is unclear. Here, we use murine models of loss of endothelial expression of HIF isoforms to explore the hypothesis that endothelial HIF-1α and HIF-2α are determinants of systemic vascular function, haemodynamics and metabolic status. We demonstrate differences between HIF deficient animals and controls in invivo and exvivo vascular and metabolic status in baseline, hypoxic and inflammatory states that suggest a role for the HIF isoforms in regulating vascular endothelial function. Methods Animal models These experimental studies were carried out under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Board (AWERB); Home Office Project License 80/2618. All animals were killed using established methods Page 3 of 14 Lambdenetal. ICMx (2021) 9:28 based on local and national guidelines derived from the Animals (Scientific Procedures) Act 1986. Mice with a tissue-specific deletion of HIF-1α in endothelial cells were created by crossing homozygous animals (C57Bl6/J) with the floxed allele in HIF-1α into a background of Cre recombinase expression driven by the Tie 2(Tek) promoter. Pulmonary endothelial deletion was driven by crossing appropriate floxed animals with mice expressing the L1 (alk-1) promoter (kindly donated by Paul Oh, University of Florida, Gainesville, FL [15]). In all experiments, animals were compared to double-floxed litter mate control mice. The number of and age of animals employed in each experiment is described in the text. Radiotelemetry All radio-telemetry hardware and software were purchased from Data Science International (MN, USA). Surgical implantation of radio-telemetry device was performed according to the manufacturer’s instructions followed by a recovery period of at least 10days. All baseline telemetry data were collected over a 72-h period in a designated quiet room which facilitates measurement of continuous haemodynamics and subcutaneous temperature. Sample size calculations were undertaken based upon previous work and designed with 80% power to detect a 10% difference in mean systolic blood pressure between knockout and control animals with an alpha of 0.05. Blood pressure monitoring during hypoxia was undertaken by combining the telemetry monitoring with Columbus Instruments Oxymax system and PEGAS mixer. Lipopolysaccharide (LPS) challenge Mice were monitored as described above using continuous telemetry for 24h prior to administration of LPS at a dose of 10mg/kg via intraperitoneal injection (Ultrapure LPS, Invitrogen). Haemodynamics and physical appearance were observed hourly after injection and culled upon reaching a predetermined humane severity endpoint. Analysis of systemic haemodynamics during baseline conditions and following LPS treatment was by unpaired t test of the area under the curve for the respective time course of each animal. Metabolic assessment Energy expenditure was measured and recorded using the Columbus Instruments Oxymax system (Columbus, OH US) according to the manufacturer’s instructions. Mice were randomly allocated to the chambers and they had free access to food and water throughout the experiment. An initial 18–24h acclimation period was disregarded for all the experiments, after which baseline data were recorded for a period of 24h. In experiments exploring the metabolic response to hypoxia, once the baseline data recording was complete, the composition of the influx gas was switched from 21% O2 to 10% O2 using a PEGAS mixer (Columbus Instruments) for 24h. Measurement ofright ventricular systolic pressure (RVSP) Mice aged 24–28weeks were weighed then anaesthetised using isoflurane at a starting dose of 2% and titrated within a 20% range to response to stimulus, heart and respiratory Page 4 of 14 Lambdenetal. ICMx (2021) 9:28 rate to determine optimum dose in each case. Catheterisation of the right side of the heart was undertaken via cannulation of the right internal jugular vein with a pressure volume loop catheter (Millar Inc, TX, USA) [16]. Measurement ofRV size Following euthanasia, the heart was removed from the thoracic cavity, and the right ventricular (RV) free wall was dissected from the left ventricle and septum (LV + S). Each portion was weighed and changes in the relative size of the RV determined by calculating the ratio RV/(LV + S) to give the Fulton index for each animal [17]. Tissue preparation In animals in whom tissue collection was undertaken the left lung was fixed using 10% (w/v) paraformaldehyde following pulmonary distension by trans-tracheal injection of 0.8% agarose. Lungs were subsequently embedded in paraffin before sectioning. Ex vivo myography Second-order femoral arteries were mounted on a four-chamber small-vessel wire myograph (Multi Wire Myograph System 610M, DMT, Denmark) [18]. Vessel normalisation was performed by determining the maximal constriction-to-diameter relationship to establish a working tension [19]. Alpha 1 adrenoreceptor-mediated constriction was evaluated in response to phenylephrine (PE, 10–10–10–4mol L−1) and tension values were corrected to the maximal response to KCL (16.4–100.9mmol L−1), as standard [8]. Relaxant responses to sodium nitroprusside (SNP) and to acetylcholine (ACh) in the range 10–8–10–4mol L−1) were determined after pre-contraction with phenylephrine (PE, 10−5mol L−1), as standard [18]. Additional concentration–response curves to ACh were determined following incubation with L-NAME (10−5mol L−1) in the same preparation. Between experiments, vessels were washed repeatedly with Krebs solution and allowed to equilibrate for at least 20min. Concentration–response curves were analysed using an agonist-response best-fit line. The contribution of NO synthase (NOS)-depend- ent mechanisms to the relaxation induced by ACh was calculated by subtracting the area under the curve (AUC) for ACh− the AUC for ACh + LNAME [20]. Blood sample analysis Whilst under anaesthesia maintained with isoflurane at an inhaled concentration of 2%, anticoagulated blood was collected from central veins by terminal exsanguination and analysed using Vet abc haematology analyser (Horiba Ltd, Japan) to determine haematological indices. Biochemical, renal and inflammatory profiling was undertaken in serum isolated from anticoagulated whole blood, which had undergone centrifugation at 1500g for 5min and frozen at –80°C. Statistical analyses The impact of genotype on systemic haemodynamics during the day–night cycle is presented as the mean (SEM) for each group over the 24-h cycle. The Student’s t test for unpaired data was used to compare control versus knockout mice using the area under the curve of the designated parameter for each animal over the 24-h cycle. Changes in Page 5 of 14 Lambdenetal. ICMx (2021) 9:28 vascular reactivity with dose and between groups were analysed by two-way repeated measures analysis of variance (RM ANOVA), and comparison of the area under the curve using the Student’s t test for unpaired data. Recovery in haemodynamics and metabolic status following the induction of hypoxia was analysed using one-phase association kinetics with trajectory of recovery curve assessed from the nadir value of the measured parameter following the onset of hypoxia and assessed over the duration of the experiment. Other data were analysed as described in the text. All analyses were undertaken using GraphPad Prism v7.04. For all statistical comparisons, significance was accepted when p < 0.05. Results Deletion ofHIF‑1α intheentire endothelium results inconstitutive cardiovascular andmetabolic dysregulation withoutimpact onthepulmonary vasculature The Tie2cre transgene deletes its genetic target at a very high rate (> 95%) in all endothelial cells, as well as in some bone marrow-derived cells [21]. Invivo haemodynamic assessment throughout the day–night cycle in mice aged 16–18weeks revealed a significantly elevated systolic blood pressure in HIF-1α deleted mice, where the transcription factor was deleted in the entire endothelial compartment with the Tie2cre transgene. In these mutant animals, mean (± SEM) systolic blood pressure was elevated relative to wild-type littermate controls (135 ± 0.99mmHg vs 115 ± 0.8mmHg, respectively; Fig. 1 Effects of HIF-1α pulmonary and systemic endothelial knockout on constitutive cardiovascular function. Circadian variations in a systolic, b diastolic blood pressure, c heart rate, d subcutaneous temperature, e oxygen consumption and f carbon dioxide synthesis of HIF-1α Tie2 Cre (green, n = 4) and littermate HIF-1α flox/flox (grey, n = 4) mice were recorded by radio-telemetry. Black box represents nocturnal phase. Haemodynamic response to 10 mg/kg LPS bolus in HIF-1α Tie2 Cre (green, n = 3) and littermate HIF-1α flox/flox (grey, n = 3) mice on g systolic and H: diastolic blood pressures and i heart rate are displayed. Data are presented as a mean ± SEM for each 30 min period, p values for area under the curve followed by unpaired t test are shown Page 6 of 14 Lambdenetal. ICMx (2021) 9:28 p = 0.006, Fig.1a). A similar pattern was seen in the diastolic blood pressures of the HIF-1α Tie2 Cre deletion mice: 98 ± 0.86mmHg versus 84 ± 0.64mmHg in wild-type littermate controls (p = 0.019, Fig.1b). No difference in heart rates was observed between in mutant and controls mice (616 ± 4 bpm vs, 606 ± 4 bpm, respectively; p = 0.86; Fig.1c). Under normoxic conditions, HIF-1α Tie2 Cre deletion was associated with significantly elevated mean ± SEM oxygen consumption compared to litter mate controls (3368 ± 62mL/min/m2 vs 2804 ± 58mL/min/m2, p = 0.019, Fig.1e) with a similar pattern of CO2 production observed (2933 ± 75 mL/min/m2 vs 2458 ± 62 mL/min/m2, p = 0.038, Fig.1f). Following the LPS challenge, the hypertensive phenotype seen under baseline conditions was preserved over the course of the period of observation with a mean ± SEM systolic blood pressure of 116 ± 1.2mmHg in knockout animals and 102 ± 2.1mmHg in litter mate controls (p = 0.006). Diastolic blood pressure showed a similar pattern, with HIF-1α Tie2 Cre mice maintaining a mean of 98 ± 0.85mmHg versus their litter mates (85 ± 0.64 mmHg, p = 0.09). In addition, whilst there was no overall difference in area under the curve for mean heart rate detected (630 ± 3.4 vs 614 ± 8.0; p = 0.534), the terminal decline in haemodynamics associated with this model was abrogated in knockout mice. Catherisation of the right hearts of mice aged 24–28weeks revealed no difference in mean ± SD ventricular systolic pressures of 25.8 ± 2.7mmHg in HIF-1α Tie2 Cre mice and 25.2 ± 1.6 mmHg in control litter mates, p = 0.778 (Additional file 1: Fig S1A). Mean ± SD Fulton index was also similar in both groups (0.1 ± 0.004 vs 0.14 ± 0.006, respectively; p = 0.78, Additional file1: Fig S1B). Pulmonary vascular histology showed similar smooth muscle development in parabronchial vessels with mean ± SD wall thickness as a proportion of mean vessel diameter of 5.22 ± 0.65% vs 6.24 ± 1.8%, p = 0.36 (Additional file 1: Fig S1C). Circumferential small-vessel muscularisation was not observed in vessels obtained from animals of either genotype (representative image Additional file1: Fig S1D). Endothelial loss ofHIF‑1α impairs endothelial function andenhances alpha‑1‑adrenoreceptor‑mediated vasoconstrictor reactivity In an exvivo analysis of femoral artery reactivity, HIF-1α Tie2 Cre mice displayed significantly impaired Acetylcholine (ACh)-induced relaxation compared to litter mate controls, p < 0.001 (Fig.2a). The impaired endothelial function in mice lacking endothelial HIF-1α was NOS-dependent. Mice lacking endothelial HIF-1α also displayed an increase in NOS-independent pathways of relaxation, however this was insufficient to normalise vascular relaxation (Fig.2b). In contrast, mice lacking endothelial HIF-1α showed normal smooth muscle-dependent dilatation in the femoral vascular bed, as sodium nitroprusside-induced relaxation was similar in both mutant and control mice (p = 0.90, Fig.2c). Mice lacking endothelial HIF-1α also showed enhanced constriction to increasing bolus doses of PE (p < 0.01, Fig.2d). Page 7 of 14 Lambdenetal. ICMx (2021) 9:28 Specific deletion ofHIF‑1α inpulmonary endothelium results insystemic haemodynamic andmetabolic dysregulation To determine how loss of HIF-1α in the endothelium within one tissue can affect systemic cardiovascular dynamics, and to control for non-endothelial effects of other knockout models, we induced deletion of HIF-1α specifically and solely in pulmonary endothelial cells. This model was developed and extensively validated as having a high degree of pulmonary specificity by the team that developed the alk1 knockout animal [15] and subsequently validated internally by our group as promoting pulmonary specific HIF gene deletion [22] In this pulmonary endothelial specific HIF-1α knockout (HIF-1α L1 Cre), a pattern of persistently raised systemic blood pressure consistent with that seen in global knockout mice was observed with both mean ± SEM systolic (126 ± 1.0mmHg vs 117 ± 0.9 mmHg, p = 0.003, Fig. 3a) and diastolic blood pressures (89.6 ± 0.97mmHg vs 86.1 ± 0.81mmHg, p = 0.005, Fig.3b) significantly elevated over the course of the day–night cycle, when compared to wild-type littermate controls. Heart rates were similar in both groups (549 ± 6bpm vs 565 ± 6bpm, p = 0.50; Fig.3c). HIF-1α L1 Cre mice were more metabolically active than their litter mate controls with VO2 and VCO2 both elevated. Mean ± SEM oxygen consumption was 3804 ± 42mL/ min/m2 in L1Cre mice versus 3193 ± 33mL/min/m2 (Fig.3e, p = 0.038) and carbon Fig. 2 Ex vivo assessment of femoral artery reactivity using wire myography in HIF-1α Tie2 Cre mice. Femoral artery reactivity was assessed using wire myography in HIF-1α Tie2 Cre (green, n = 5) and HIF-1α flox/flox littermates (grey, n = 5). Data presented as Mean ± SEM. For concentration response curves, analysis was by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). The NOS-dependent/independent components expressed as area under the curve were analysed by One-way ANOVA with Tukey test (*p < 0.05 vs. NOS-dependent, #p < 0.05 vs. NOS-independent, †p < 0.05 vs. total effect). Data for the KCl, Phe and SNP area under the curve were analysed by the Student’s t test for unpaired data (*p < 0.05). a Degree of relaxation was expressed as a percentage of the contraction induced by phenylephrine. [ACh]: molar concentration of acetylcholine. b The contribution of NO-independent mechanisms was calculated by the AUC for ACh + LNAME (10−5 M), *p < 0.05 for NO (Nitric Oxide) dependent and # p < 0.05 for NO-independent portions of ACh-induced relaxation. c Degree of relaxation was expressed as a percentage of the contraction induced by phenylephrine. [SNP]: molar concentration of sodium nitroprusside and d degree of vasoconstriction developed in response to increasing molar concentrations of phenylephrine ([Phe] 10−5 M) Page 8 of 14 Lambdenetal. ICMx (2021) 9:28 dioxide production was 3484 ± 49mL/min/m2 in knockouts vs 2960 ± 38mL/min/m2 in wild-type mice (Fig.3f, p = 0.03). When right ventricular systolic pressures were assessed, a mean ± SD RVSP of 23.2 ± 2.33mmHg was observed in pulmonary endothelial HIF-1α null animals, and 23.2 ± 2.22mmHg in wild-type controls (p = 0.99, Additional file1: Fig S1E). The Fulton index was 0.163 ± 0.02 in HIF-1α L1 Cre pulmonary endothelial deletion mice vs 0.156 ± 0.03 in wild-type control mice (p = 0.66, Additional file1: Fig S1F). No differences were observed in parabronchial blood vessel smooth muscle thickness as a proportion of average diameter, with mean (SD) wall thickness 6.28 ± 2.0% in L1 Cre mice and 7.2 ± 1.9% in wild-type littermates, p = 0.46, (Additional file1: Fig S1G; representative images of SMA, vWF, and H&E staining are shown in Additional file1: Fig S1Hi, 1Hii, 1Hiii, respectively). No circumferential small-vessel muscularisation was seen in mice of either genotype (representative image Additional file1: Fig S1Hiv). The haematological, biochemical, and inflammatory profiles of 24–28week-old pulmonary endothelial HIF-1α null mice and wild-type littermate controls were compared. No significant differences in haemoglobin (Additional file1: Fig S2A) or red blood cell count (Additional file1: Fig S2B) were seen under normoxic conditions. HIF-1α L1 Fig. 3 Effects of HIF-1α pulmonary endothelial knockout on constitutive cardiovascular function. Circadian variations in a systolic, b diastolic blood pressure, c heart rate, d subcutaneous temperature, e VO2 and f VCO2 of HIF-1α L1 Cre (red, n = 11) and littermate HIF-1α flox/flox (grey, n = 9) mice were recorded by radio-telemetry. Black box represents nocturnal phase. Data are presented as a mean ± SEM for each 30 min period, p value for area under the curve followed by unpaired t test are shown. Femoral artery reactivity was assessed using wire myography in HIF-1α L1Cre (red, n = 5) and HIF-1α flox/flox littermates (grey, n = 5). g Degree of relaxation was expressed as a percentage of the contraction induced by phenylephrine. [ACh]: molar concentration of acetylcholine. h Degree of relaxation was expressed as a percentage of the contraction induced by phenylephrine. [SNP]: molar concentration of sodium nitroprusside and i degree of vasoconstriction developed in response to increasing molar concentrations of phenylephrine ([Phe] 10−5 M). Data presented as Mean ± SEM for concentration response, analysis by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001) Page 9 of 14 Lambdenetal. ICMx (2021) 9:28 Cre pulmonary endothelial deletion animals did not display significant differences in systemic markers of renal function (Additional file1: Fig S2C) or serum electrolytes (Additional file1: Fig S2D) when compared to litter mate controls. No differences in inflammatory state were detected across a panel of ten biomarkers (Additional file1: Fig S2E and 2F). The impact ofpulmonary endothelial HIF‑1α knockout onsystemic vascular function HIF-1α L1 Cre pulmonary endothelial deletion mice aged 24–28weeks also displayed impaired femoral artery relaxation to ACh, when compared to controls. However, the ACh-mediated relaxation was not only abolished, but was also reversed to constriction, p < 0.001 (Fig.3g). SNP-induced vasodilatation was significantly impaired (p < 0.01, Fig.3h) and PE-induced constriction exaggerated in HIF-1-deficient mice compared to controls (p < 0.01, Fig.3i). Loss ofpulmonary endothelial HIF‑2α isnotassociated withbaseline haemodynamic ormetabolic abnormality The HIF-2α L1Cre mice displayed no significant differences in systemic haemodynamics or metabolic status under normoxic conditions. Mean ± SEM systolic blood pressures (119 ± 1.2mmHg versus 117 ± 1.1mmHg, p = 0.76 Additional file1: Fig S3A), diastolic blood pressures (84 ± 1.0mmHg vs 85 ± 0.9mmHg, p = 0.6 Additional file1: Fig S3B), heart rate (577 ± 8bpm vs 583 ± 7bpm, p = 0.79 Additional file1: Fig S3C) and subcutaneous temperature (33.5 ± 0.1°C vs 32.8 ± 0.1°C, p = 0.35 Additional file1: Fig S3D) were all similar in knockout and wild-type littermates, respectively. Mean ± SEM oxygen consumption was 3598 ± 97mL/min/m2 in HIF-2α L1Cre mice and 3604 ± 75 mL/min/m2 in litter mate controls, p = 0.98 (Additional file1: Fig S3E). VCO2 was 3313 ± 105mL/min/m2 in knockout mice and 3326 ± 96mL/min/m2 in controls, p = 0.95 (Additional file1: Fig S3F). There was no change in pulmonary vascular wall thickness in the animals deficient in HIF-2α at baseline (p = 0.45, Additional file1: Fig S3G). Loss ofHIF isoforms inpulmonary endothelium modulates thesystemic cardiovascular andmetabolic toenvironmental hypoxia andacute inflammation HIF-1α L1 Cre mice and litter mate controls were exposed to 10% oxygen (hypoxia) following a 48-h period of environmental adaptation to the metabolic chamber. Wildtype control mice have a triphasic response to acute hypoxia, characterised by a short initial tachycardia and hypertension, followed by a rapid reduction in both heart rate and blood pressure [23]. This is followed by a partial-to-complete recovery after 24 to 36h. One-phase association curve fitting was used to analyse the recovery phase following the onset of hypoxia. HIF-1α-deficient mice displayed similar systolic blood pressure responses to their litter mates when exposed to acute hypoxia (Fig.4a). However, a lower nadir and slower rate of recovery of diastolic blood pressure was seen following the onset of hypoxia, compared to litter mates (Fig.4b, p < 0.0001). Whilst heart rate responses were similar in both groups (Fig.4c), subcutaneous temperature, an indirect indicator of the vascular resistance within the skin [24], displayed a similar pattern to