Published online before print August 14, 2008, doi: 10.1161/CIRCRESAHA.108.182758
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Integrative Physiology |
Chronic Hypoxia–Induced Angiogenesis Normalizes Blood Pressure in Spontaneously Hypertensive Rats
From the Cardiovascular Research Center, Institut National de la Santé et de la Recherche Médicale Lariboisière Unit 689, and Université Paris 7-Denis Diderot, Hôpital Lariboisière, Paris, France.
Correspondence to Bernard Lévy, U689 INSERM, Hôpital Lariboisière, 41 Bd de la Chapelle, 75475 Paris Cedex 10, France. E-mail bernard.levy{at}inserm.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We hypothesized that activation of angiogenesis by chronic hypoxia may affect vascular resistance and, subsequently, blood pressure levels in spontaneously hypertensive rats (SHRs). Five-week-old prehypertensive SHRs and age-matched normotensive Wistar–Kyoto (WKY) rats (n=8 per group) were maintained under normobaric normoxic or hypoxic (10% O2) conditions for 8 weeks. Three weeks later, the systolic blood pressure was lower by 26% in hypoxic SHRs compared to normoxic SHRs (P<0.05) and remained at the normoxic WKY level. Total peripheral vascular resistance, calculated as the mean arterial pressure/cardiac output (assessed by ultrasound imaging and Doppler), was 30% lower in hypoxic than in normoxic SHRs (P<0.001) and returned to WKY levels. Interestingly, chronic hypoxia also significantly reduced systolic blood pressure in adult 12-week-old SHRs with established hypertension; blood pressure was normalized (versus normoxic WKY rats) after 4 weeks of hypoxia. Changes in hemodynamic parameters were associated with activation of proangiogenic pathways. Protein levels of vascular endothelial growth factor (VEGF)-A in the skeletal muscles were increased by 2.2-fold in hypoxic compared to normoxic SHRs (P<0.001). At the end of the hypoxic period, capillary density in the quadriceps muscle was 1.2-fold higher in hypoxic than in normoxic SHRs (P<0.001). Myocardial capillary density and VEGF-A protein contents were also 1.2- and 2.1-fold higher in hypoxic compared to normoxic SHRs (P<0.001 and P<0.05, respectively). Moreover, treatment with neutralizing VEGF-A antibody abrogated the hypoxia-induced angiogenesis and subsequently worsened arterial hypertension. Therefore, our results suggest that chronic normobaric hypoxia (1) activates VEGF-A–induced angiogenesis and thereafter (2) prevents the occurrence of hypertension in young prehypertensive SHRs and (3) normalizes blood pressure in adult SHRs with established hypertension.
Key Words: hypoxia • angiogenesis • hypertension
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In most forms of clinical and experimental hypertension, increased arterial blood pressure is associated with microvascular rarefaction and upregulation of peripheral vascular resistances.1 The cause-and-effect relationships of vascular rarefaction and hypertension are still debated. It is speculated that diffuse systemic rarefaction might be a primary defect in essential hypertension.2 On the other hand, rarefaction can also represent a downstream consequence, as shown by its occurrence in animal models of secondary hypertension.3,4 Microvascular density might decrease because of either vessel destruction or insufficient angiogenesis, ie, growth of new capillaries from preexisting ones. This process proceeds during development and also in adults during physiological and pathological conditions. Abnormally low microvascular density can be seen at a very young age in animals with genetic hypertension5 and also exists in normotensive humans with a familial predisposition to the disease,6,7 indicating that alteration in vessel growth could lead to elevation in the peripheral vascular resistance and subsequently trigger the pathogenesis of hypertension.2 Hence, one can speculate that changes in proangiogenic related pathways may directly affect blood pressure levels.
Interestingly, epidemiological studies have shown that humans living at high altitude have lower systemic blood pressures than those living at the sea level.8–10 Although some metabolic11 or hormonal12 modifications have been described, the exact mechanism by which hypoxia prevents the development of hypertension remains largely undefined. We therefore hypothesized that hypoxia may trigger angiogenesis, resulting in reduction of blood pressure levels. Indeed, hypoxia is known to trigger angiogenesis. The main mechanism of hypoxia-induced capillary growth involves the rise in hypoxia-inducible factor (HIF)-1 protein. HIF-1 binds to specific hypoxia-responsive element in the regulatory regions of several hypoxia-sensitive genes, such as vascular endothelial growth factor (VEGF)-A.13 VEGF-A is then secreted and binds to its cognate receptor tyrosine kinases, Flt-1 and Flk-1/KDR, located on the surface of vascular cells. Receptor ligation triggers a cascade of intracellular signaling pathways that initiate angiogenesis. VEGF-A has been shown, through Flk-1/KDR, to activate endothelial nitric oxide synthase (eNOS)-related pathways, leading to nitric oxide (NO) production.14
To unravel the link between hypertension and angiogenesis, we analyzed the effect of normobaric hypoxia on blood pressure levels in animals with genetic hypertension. We showed that chronic hypoxia (1) activates VEGF-A–induced angiogenesis and thereafter (2) prevents the occurrence of hypertension in young prehypertensive SHRs and (3) normalizes blood pressure in adult SHRs with established hypertension.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental Design
Experiments were conducted according to the French veterinary guidelines and those formulated by the European Community for experimental animal use. All rats were obtained from Charles River (Lyon, France).
Experiment 1
Prehypertensive young SHRs (5 weeks old) treated with or without VEGF-A–neutralizing antibody (3 mg/kg IP, twice a week; Genentech, San Francisco, Calif) and age-matched Wistar–Kyoto (WKY) rats were housed for 3 or 7 weeks under normoxic (standard laboratory conditions, ie, 20.5% O2) or hypoxic (10% O2) conditions (n=8 per group). Hypoxic (normobaric) conditions were obtained in a hypoxic chamber (Coy Laboratories), maintained at 25°C and 80% humidity. CO2 production by the animals was fixed with soda lime and PCO2 remained at the 0 level in the hypoxic chamber.
Experiment 2
Hypertensive adult SHRs (12 weeks old) were housed under normobaric hypoxic conditions for 8 weeks and then under normoxic conditions for 2 hours or 3 additional weeks before euthanasia. In additional set of experiments, hypertensive adult SHRs (12 weeks old) were housed under hypoxic conditions for 1 week and then under normoxic conditions for 1 additional week before euthanasia. Systolic blood pressure (SBP) was measured weekly in all young and adult conscious rats using a computerized tail-cuff system (BP2000 Visitech Systems).15
Ultrasonic Measurements
Anesthetized rats (1.5% isoflurane in 100% O2) were maintained in left lateral decubitus position. The pulmonary artery diameter was then measured between the pulmonary sigmoid valves, using an ultrasound echocardiograph (Vivid 7, GE Medical Systems, Horten, Norway) equipped with a 12-MHz linear transducer. The blood flow was also recorded at the level of the sigmoid valves. Heart rate, peak systolic, and time-averaged mean blood flow velocity were measured with correction of the angle between the long axis of the pulmonary artery and the Doppler beam axis. Calculation of Cardiac Index (CI) (milliliters per minute) was as follow: CI=60x{[Vmean]x[
x(Dpa/2)2]}. Vmean is the time-averaged mean blood flow velocity (in centimeters per second) and Dpa is the pulmonary artery internal diameter (in centimeters). Total peripheral resistance (TPR) was then calculated as MBP/COs; MBP is the mean arterial blood pressure, and CIs is the cardiac output normalized to the body surface [body surface (cm2)=9.1xbody weight0.66 (g)16].
Microangiography
Vessel density was evaluated by high-definition microangiography at the end of the treatment period, as previously described.17 Briefly, animals were anesthetized (isoflurane inhalation) and a contrast medium (barium sulfate, 5 g/mL) was injected through a catheter introduced into the abdominal aorta. Images acquired by a digital x-ray transducer were assembled to obtain a complete view of hindlimbs. The angiographic score was expressed as a percentage of pixels per image occupied by vessels in the quantification area. Quantification zone was delimitated by the knee, the edge of the femur, and the external limit of the leg.
Capillary and Arteriole Density
Quadriceps muscle and the heart were dissected and progressively frozen in isopentane solution precooled in liquid nitrogen. Frozen tissue sections (7 µm) were incubated with rabbit polyclonal antibody directed against total fibronectin (dilution 1:50; Chemicon International) to identify capillaries or with mouse monoclonal antibody directed against human smooth muscle actin 
-1 (dilution 1/50; Dako Cytomation) to identify arterioles. Vessel density was calculated in 10 randomly chosen fields (300 to 500 µm2) using Histolab software (Microvision, Evry, France).
Reverse Transcription and Quantitative Real-Time PCR
Total RNA was isolated from quadriceps muscle or heart according to the TRIzol reagent protocol (Invitrogen). One microgram of total RNA was reverse-transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen). Real-time PCRs were performed with a GeneAmp 9700 (Applied Systems) in a total volume of 25 µL using Platinum SYBR Green qPCR kit (Invitrogen) under the following conditions: 2 minutes at 50°C and 2 minutes at 95°C, followed by a total of 40 cycles of 3 temperature cycles (15 seconds at 95°C, 15 seconds at annealing temperature, see primer compositions below, and 20 seconds at 72°C). To confirm amplification specificity, the PCR products from each primer pair were subjected to melting curve analysis and subsequent agarose gel electrophoresis. Each sample was analyzed in triplicate and the relative expression for each amplicon was calculated by a mathematical method based on the real-time PCR efficiencies18 using GAPDH as reference. The primers and annealing temperatures were as follow: VEGF-A forward, 5'-CGACAGAAGGGGAGCAGAAAGC-3'; VEGF-A reverse, 5'-CGCACACCGCATTAGGGGCACA-3' (annealing 61.6°C); FLK-1 forward, 5'-GGCGAATCACTCACACCAGTT-3'; FLK-1 reverse, 5'-CTCATCCAAGGGCAGTTCATCT-3' (annealing 60.5°C); HIF-1 forward, 5'-CAGCGATATGGTCAATGTATTCAAGT-3'; HIF-1 reverse, 5'-TGGCAGTGACAGTGATGGTAGG-3' (annealing 58.4°C); GAPDH forward, 5'-AGTGCCAGCCTCGTCTCATA-3'; GAPDH reverse, 5'-CTTGCCGTGGGTAGAGTCATAC-3' (annealing 59.4°C).
Western Blot
Tissue samples (quadriceps muscle and heart) were thawed and homogenized in 500 µL of radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mmol/L Tris, pH 7.4) containing protease inhibitors (Roche). Protein content was then determined by the method of Lowry adapted for SDS containing samples (Bio-Rad). Proteins were separated in denaturing SDS 9% polyacrylamide gels and then blotted onto nitrocellulose sheet (Bio-Rad). Antibodies against VEGF-A (1:2000; Santa Cruz Biotechnology), eNOS (1:1000; Santa Cruz Biotechnology), and FLK-1 (1:1000; US Biological) were used. After the immunoblots were washed, secondary antibody conjugated to horseradish peroxidase (Amersham Life Sciences) was added and developed using ECL detection kit (Amersham Life Sciences). As a protein loading control, membranes were stripped and incubated with a monoclonal antibody directed against total actin (1:5000; Sigma). Chemiluminescent signals were then acquired using a Fujifilm LAS-1000 imager and densitometric analyses were performed with ImageJ software (NIH).
Statistics
Results are expressed as means±SEM. Two-way ANOVA was used to compare each parameter. Post hoc Bonferroni’s test comparisons were then performed to identify which group differences account for the significant overall ANOVA. Student’s t test was used to compare hypoxic SHRs treated with or without VEGF-A–neutralizing antibody. A value of P<0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effect of Hypoxia on Morphological Parameters
In WKY rats, hypoxia decreased the body weight by 30% compared to normoxic WKY rats (P<0.001, Table). The heart-to-body weight ratio was raised by 20% in hypoxic WKY rats in reference to normoxic WKY rats (P<0.01), likely reflecting the lower body weight and the right ventricular hypertrophy associated with hypoxia. In SHRs, body weight was reduced by 40% under hypoxic condition (P<0.001), whereas the heart to body weight ratio was increased by 20% in hypoxic compared to normoxic SHRs (P<0.001). In both hypoxic and normoxic conditions, body weight was lower in SHRs compared to WKY rats (Table). The heart-to-body weight ratio was higher by
20% in SHRs compared to WKY rats, regardless of the experimental conditions.
|
Effect of Hypoxia on Hemodynamic Parameters
We first assessed the effect of hypoxia on the development of hypertension in prehypertensive SHRs. Prehypertensive young SHRs, maintained under normoxic conditions, showed higher SBP than normoxic WKY rats, throughout the experiment (Figure 1A). In WKY rats, hypoxia tended to reduce SBP when compared to normoxic WKY rats; this difference reached significance after 3 weeks of hypoxia. In SHRs, after 3 weeks of exposure to hypoxia, SBP was lower by 26% in hypoxic SHRs compared to normoxic SHRs (P<0.05). Thereafter, SBP remained lower in hypoxic SHRs compared to normoxic SHRs and was similar to the SBP in normoxic WKY rats during the last 4 weeks of hypoxic period. Cardiac output was not affected by hypoxia in both WKY and SHR strains. Changes in SBP were thus associated with modifications in TPR. TPR was raised by 30% in normoxic SHRs compared to normoxic WKY rats (P<0.001). In hypoxic SHRs, TPR was reduced by 30% when compared to normoxic SHRs (P<0.001) and returned to WKY levels (Table). In WKY rats, hypoxia tended to reduce TPR, but this difference did not reach statistical significance. Taken together, these results suggest that hypoxia decreased TPR, preventing the rise in SBP occurring in normoxic SHRs.
|
P<0.05, We next analyzed the effect of hypoxia in adult SHRs with established hypertension (Figure 1B). Interestingly, SBP in adult hypoxic SHRs was also reduced by 16% (P<0.001, versus normoxic SHRs) as early as 1 week of hypoxia, suggesting that hypoxia reversed hypertension in adult SHRs. In addition, after 8 weeks of hypoxia, return to normoxia was followed by increased SBP in both 5- and 12-week-old SHRs. Taken together, these results suggest that (1) hypoxia was able to prevent the raise in blood pressure levels or to reverse hypertension and (2) this hypoxia-related effect was transient and fully abrogated after return to normoxia.
Mechanisms of Hypoxia-Induced Reduction in SBP in SHRs
We next attempted to understand the molecular and cellular mechanisms associated with the hypoxia-induced prevention of hypertension development in young SHRs. Hypoxia is a well-known activator of angiogenesis.13 We therefore analyzed the effect of hypoxia on blood vessel growth.
We first analyzed the density of vascular structure in the hindlimb of hypoxic and normoxic animals. Microangiography analysis indicated that arteriole (>200 µm) density was similar in normoxic WKY rats and normoxic SHRs. In addition, hypoxia did not significantly modify arterial angiographic score in the hindlimb of WKY rats and SHRs (Figure 2A). In parallel, arteriole density (measured in skeletal muscle histological sections) was similar in WKY rats and SHRs, regardless of the conditions (Figure 2B). In contrast, skeletal muscle capillary density was decreased by 30% in normoxic SHRs compared to normoxic WKY rats (P<0.01). Interestingly, hypoxia raised capillary density by 30% in both hypoxic SHRs and WKY rats compared to their normoxic control animals (Figure 3A). Capillary density in hypoxic SHRs was then similar to that in normoxic WKY rats.
|
|
We analyzed the effect of hypoxia in the left ventricle as well. We did not observe any significant changes in arteriole density in the left ventricle free wall from WKY rats and SHRs, regardless of the experimental conditions. In contrast, capillary density was lower in normoxic SHRs compared to normoxic WKY rats (P<0.05). Capillary density was increased by 20% in hypoxic SHRs compared to normoxic SHRs (P<0.001) and was similar to that of WKY rats.
Finally, return to normoxia for 2 hours did not reverse the reduction in blood pressure observed in hypoxic SHRs (Figure 4A). This does not support a short-term effect of hypoxia-induced vasoactive substances (especially of VEGF, potent vasodilator peptide) but is, rather, in favor of an effect of the structure of the vascular network on blood pressure. Nevertheless, changes in blood pressure levels and capillary number were fast. Indeed, we performed additional experiments to analyze the capillary densities in SHRs maintained for 1 week in hypoxia followed or not by 1 additional week under normoxic conditions. We showed that 1 week of hypoxia decreased by 27% the blood pressure levels and increased capillary number by 1.4-fold in hypoxic SHRs compared to normoxic SHRs. Interestingly, these effects were blunted after return to normoxia for one week (Figure 4B and 4C). All together, these results suggest that hypoxia triggers angiogenesis within the skeletal muscles and the myocardium, indicating that hypoxia-induced increase in capillary density may reduce TPR and thus SBP in the SHRs.
|
It is also noteworthy that we did not observe any significant differences in the in vivo and in situ basal and sodium nitroprusside-induced maximal diameters of mesenteric arteries (80 to 150 µm) in normoxic and hypoxic SHRs, suggesting that hypoxia did not affect the basal diameter of resistance arteries and did not modify their maximal dilation (absence of arteriole remodeling) (Figure I in the online data supplement, available at http://circres. ahajournals.org).
Molecular Mechanisms of Hypoxia-Induced Angiogenesis
We next sought to identify the molecular mechanisms involved in hypoxia-induced angiogenesis. We analyzed the regulations of key genes involved in hypoxia-induced angiogenesis, such as HIF-1, VEGF-A, its receptor Flk-1/KDR, and eNOS.13
We did not observed any significant differences in HIF-1 mRNA levels in our experimental conditions (Figure 5A and 5B). VEGF-A mRNA expression was reduced by 50% in the normoxic SHR hindlimb compared to normoxic WKY rats. Interestingly, hypoxia increased VEGF-A mRNA contents in both hindlimb and heart of hypoxic SHRs compared to normoxic SHRs (Figure 5A and 5B). Similarly, Flk-1 mRNA levels were also upregulated in both hindlimb and heart of hypoxic SHRs compared to normoxic SHRs (Figure 5A and 5B).
|
Changes in mRNA levels were associated with modifications in protein levels. VEGF-A protein levels were decreased by 54% and 43% in the hindlimb and heart, respectively, in normoxic SHRs compared to normoxic WKY rats (Figure 6A and 6B). Hypoxia increased VEGF-A content by 2.2- and 2.1-fold in the hindlimb and heart, respectively, of hypoxic SHRs compared to normoxic SHRs (P<0.01). Subsequently, VEGF-A protein levels in hypoxic SHRs returned to normoxic WKY levels. VEGF-A receptor Flk-1 protein contents were also reduced by 42% in the hindlimb of normoxic SHRs compared to normoxic WKY rats (P<0.01). Hypoxia upregulated Flk-1 contents by 2.4-fold in the hindlimb of hypoxic SHRs compared to normoxic SHRs (P<0.01). Subsequently, Flk-1 protein levels in hypoxic SHRs returned to normoxic WKY levels. In contrast, Flk-1 protein content in heart was similar between normoxic SHRs and normoxic WKY rats and was not modulated by hypoxia in SHRs. Finally, cotreatment with VEGF-A–neutralizing antibody fully abrogated the hypoxia-induced angiogenesis in young prehypertensive SHRs and restored high blood pressure levels in treated hypoxic SHRs (Figure 7).
|
|
All together, our results underlined that VEGF-A/Flk-1–related pathway is involved in hypoxia-induced angiogenesis in hypertensive animals.
Finally, eNOS protein levels were not modified by hypoxia in SHRs (Figure 6A and 6B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The main result of this study is that hypoxia blunts the development of hypertension in young prehypertensive SHRs and reverses hypertension in adult SHRs with established hypertension. Activation of VEGF-A–dependent angiogenesis plays a major role in these hypoxia-induced modifications of blood pressure levels.
Several authors have previously assessed the effect of hypoxia on hemodynamic parameters in the SHRs. Most were interested by transient pulmonary hypertension.19 Henley and Tucker reported that chronic hypoxia resulted in decrease systemic blood pressure in young SHRs. Although thyroid status was unchanged, the authors suggested that thyroid hormones may be involved in these hypoxia-related antihypertensive effect.20 Alternatively, the lowering of the arterial pressure could depend on central effects of the hypoxia on the center of the arterial pressure regulation.21 However, the effects of hypoxia on blood pressure are believed to result in sympathetic activity increase, leading to upregulation of blood pressure and heart rate.22 Our results also show that hypoxia did not affect cardiac output but decreased TPR and subsequently blood pressure levels. Therefore, the mechanisms underlying the effect of hypoxia on blood pressure levels remained unresolved. It is widely admitted that microvascular rarefaction contributes to the increase of TPR in hypertension.1 In support of this view, we suggest, in the present work, that changes in TPR are correlated with modifications of tissue capillary densities. Hence, hypoxia-induced increase in capillary density could reduce TPR and prevent the development of hypertension in SHRs or reduce blood pressure levels in established hypertensive SHRs. Therefore, changes in capillary density might be a key factor involved in the control of blood pressure levels. Previous studies support a primary role for capillary rarefaction in the development of hypertension. In human subjects, skin capillary rarefaction has been reported in normotensive young adults with a genetic propensity to develop high blood pressure.6,23 Microvascular rarefaction can be detected in patients with only mild or borderline hypertension24 and progresses in parallel with the severity of hypertension. In addition, antihypertensive drug treatments increase capillary density in hypertensive subjects.25
The effect of hypoxia on blood pressure levels is likely related to activation of VEGF-A–dependent angiogenesis. Hypoxia is a well-known stimulus for angiogenesis through activation of HIF-1 signaling.13 Although increased levels of HIF-1 mRNA have been reported, most of the studies suggest that HIF-1 is mainly regulated at the translational or posttranslational levels.26 However, HIF-1 protein contents rapidly undergo proteosomal degradation.27 Accordingly, we did not find evidence of significant changes in HIF-1 mRNA levels. In contrast, we showed that hypoxia highly stimulates expression of HIF-1–related genes, VEGF-A, and its receptor, Flk-1. Previous reports on the influence of hypoxia on Flk-1 expression are controversial.28–31 Nevertheless, VEGF-A gene transfer restored Flk-1 mRNA levels in a rat sponge model, suggesting that hypoxia-induced VEGF-A upregulation may enhance Flk-1 contents.32 Activation of VEGF-A–related pathways has been shown to promote endothelial cells migration, proliferation, survival, and proteolytic activity and may thereby activate angiogenesis. Interestingly, cotreatment with VEGF-A–neutralizing antibody blocked hypoxia-related effects on angiogenesis and blood pressure levels. It is also noteworthy that VEGF-A protein contents are reduced in normoxic SHRs compared to normoxic WKY rats, suggesting that the decrease in VEGF-A levels could be involved in capillary rarefaction in this setting. In addition, we and others have previously shown that antihypertensive agents, such as angiotensin-converting enzyme inhibitor, raise VEGF-A levels and promote angiogenesis.33,34 Finally, severe hypertension is more common (up to 38%) in patients with metastatic colorectal cancer with monoclonal antibody against VEGF-A.35 We have recently reported that pharmacological blockade of VEGF with bevacizumab (VEGF antibody) in patients with colorectal cancer resulted in significant capillary rarefaction and parallel increase in blood pressure. Both changes were closely associated with and seemed at least partially responsible for the rise in arterial pressure.36 All together, these results suggest that VEGF-A–dependent angiogenesis controls blood pressure levels in adults.
However, we cannot preclude the hypothesis that part of the anti-VEGF antibody–related effects might be related to the multiple actions of VEGF-A on vascular function. In this view, VEGF-A induces endothelium-dependent vasodilatation in normotensive and hypertensive conditions.37,38 However, this hypotensive effect was only detected after acute infusion of VEGF-A (2 hours) or when high doses of VEGF-A were chronically infused (7 days). In an additional set of experiments, we observed that return to normoxia for 2 hours did not reverse the reduction in blood pressure observed in hypoxic SHRs, suggesting that changes in blood pressure were not associated with the acute vasoactive effects of VEGF-A but rather with its structural proangiogenic actions.
In conclusion, this study shows that hypoxia-induced angiogenesis prevents the microvascular rarefaction, which normally occurs in the course of hypertension and subsequently abrogates the hypertensive status in SHRs. Therefore, therapeutic strategies designed to improve tissue angiogenesis may affect blood pressure levels and may constitute a promising therapeutic avenue in the treatment of hypertension.
|
Acknowledgments |
|---|
We thank Genentech (San Francisco, Calif) for the kind gift of rat anti-VEGF antibody.
Sources of Funding
J.-S.S. is supported by Agence Nationale de Recherches "Young Investigator" grant JC05-45445 and Agence Nationale de Recherches "Cardiovascular, Obesity and Diabetes" grants Agence Nationale de la Recherche-05-028-01, ANR-05-022-02. Institut National de la Santé et de la Recherche Médicale U689 is a partner of the European Vascular Genomics Network (EVGN), a Network of Excellence of the European Commission (contract no. LSHM-CT-2003-503254). We thank Institut National de la Santé et de la Recherche Médicale, Université Paris 7, Naturalia & Biologia, and Société pour le Dévelopement de la Recherche Cardiovasculaire for their support.
Disclosures
None.
|
Footnotes |
|---|
Original received May 14, 2007; resubmission received July 8, 2008; revised resubmission received August 4, 2008; accepted August 6, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Feihl F, Liaudet L, Waeber B, Levy BI. Hypertension: a disease of the microcirculation? Hypertension. 2006; 48: 1012–1017.
2. Levy BI, Ambrosio G, Pries AR, Struijker-Boudier HA. Microcirculation in hypertension: a new target for treatment? Circulation. 2001; 104: 735–740.
3. Levy BI, Duriez M, Samuel JL. Coronary microvasculature alteration in hypertensive rats. Effect of treatment with a diuretic and an ACE inhibitor. Am J Hypertens. 2001; 14: 7–13.[CrossRef][Medline] [Order article via Infotrieve]
4. Sabri A, Samuel JL, Marotte F, Poitevin P, Rappaport L, Levy BI. Microvasculature in angiotensin II-dependent cardiac hypertrophy in the rat. Hypertension. 1998; 32: 371–375.
5. Chen II, Prewitt RL, Dowell RF. Microvascular rarefaction in spontaneously hypertensive rat cremaster muscle. Am J Physiol. 1981; 241: H306–H310.[Medline] [Order article via Infotrieve]
6. Antonios TF, Rattray FM, Singer DR, Markandu ND, Mortimer PS, MacGregor GA. Rarefaction of skin capillaries in normotensive offspring of individuals with essential hypertension. Heart. 2003; 89: 175–178.
7. Noon JP, Walker BR, Webb DJ, Shore AC, Holton DW, Edwards HV, Watt GC. Impaired microvascular dilatation and capillary rarefaction in young adults with a predisposition to high blood pressure. J Clin Invest. 1997; 99: 1873–1879.[Medline] [Order article via Infotrieve]
8. Lei S, Yong-Yong X, Xiao-Han D, Chang-Sheng C. Geographical differences in blood pressure of male youth aged 17–21 years in China. Blood Press. 2004; 13: 169–175.[CrossRef][Medline] [Order article via Infotrieve]
9. Rotta A. Physiologic condition of the heart in the natives of high altitude. Am Heart J. 1947; 33: 669–676.[CrossRef]
10. Ruiz L, Penaloza D. Altitude and hypertension. Mayo Clin Proc. 1977; 52: 442–445.[Medline] [Order article via Infotrieve]
11. Behm R, Honig A, Griethe M, Schmidt M, Schneider P. Sustained suppression of voluntary sodium intake of spontaneously hypertensive rats (SHR) in hypobaric hypoxia. Biomed Biochim Acta. 1984; 43: 975–985.[Medline] [Order article via Infotrieve]
12. Barbarash NA, Dvurechenskaia G, Volina EV, Berdysheva LV, Putintseva TG. [Effect of adaptation to the altitude hypoxia on catecholamine metabolism in rats with spontaneous hereditary hypertension]. Biull Eksp Biol Med. 1982; 93: 22–24.[Medline] [Order article via Infotrieve]
13. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003; 9: 653–660.[CrossRef][Medline] [Order article via Infotrieve]
14. Blanes MG, Oubaha M, Rautureau Y, Gratton JP. Phosphorylation of tyrosine 801 of the VEGFR-2 is necessary for AKT-dependent eNOS activation and nitric oxide release from endothelial cells. J Biol Chem. 2007; 282: 10660–10669.
15. Krege JH, Hodgin JB, Hagaman JR, Smithies O. A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension. 1995; 25: 1111–1115.
16. Baker HJ, Lindsey JR, Weisbroth SH. Biology and diseases. In: Baker HJ, Lindsey JR, Weisbroth SH, eds. The Laboratory Rat. New York: Academic Press; 1979, 59–74.
17. Silvestre JS, Mallat Z, Tamarat R, Duriez M, Tedgui A, Levy BI. Regulation of matrix metalloproteinase activity in ischemic tissue by interleukin-10: role in ischemia-induced angiogenesis. Circ Res. 2001; 89: 259–264.
18. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29: e45.
19. Janssens SP, Thompson BT, Spence CR, Hales CA. Functional and structural changes with hypoxia in pulmonary circulation of spontaneously hypertensive rats. J Appl Physiol. 1994; 77: 1101–1107.
20. Henley WN, Tucker A. Hypoxic moderation of systemic hypertension in the spontaneously hypertensive rat. Am J Physiol. 1987; 252: R554–R561.[Medline] [Order article via Infotrieve]
21. Mifflin S. New insights into the electrophysiology of brainstem circuits controlling blood pressure. Curr Hypertens Rep. 2007; 9: 236–241.[CrossRef][Medline] [Order article via Infotrieve]
22. Hainsworth R, Drinkhill MJ, Rivera-Chira M. The autonomic nervous system at high altitude. Clin Auton Res. 2007; 17: 13–19.[CrossRef][Medline] [Order article via Infotrieve]
23. Sullivan JM, Prewitt RL, Josephs JA. Attenuation of the microcirculation in young patients with high-output borderline hypertension. Hypertension. 1983; 5: 844–851.
24. Gasser P, Buhler FR. Nailfold microcirculation in normotensive and essential hypertensive subjects, as assessed by video-microscopy. J Hypertens. 1992; 10: 83–86.[CrossRef][Medline] [Order article via Infotrieve]
25. Debbabi H, Uzan L, Mourad JJ, Safar M, Levy BI, Tibirica E. Increased skin capillary density in treated essential hypertensive patients. Am J Hypertens. 2006; 19: 477–483.[CrossRef][Medline] [Order article via Infotrieve]
26. Gleadle JM, Ratcliffe PJ. Hypoxia and the regulation of gene expression. Mol Med Today. 1998; 4: 122–129.[CrossRef][Medline] [Order article via Infotrieve]
27. Berra E, Ginouves A, Pouyssegur J. The hypoxia-inducible-factor hydroxylases bring fresh air into hypoxia signalling. EMBO Rep. 2006; 7: 41–45.[CrossRef][Medline] [Order article via Infotrieve]
28. Brogi E, Schatteman G, Wu T, Kim EA, Varticovski L, Keyt B, Isner JM. Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. J Clin Invest. 1996; 97: 469–476.[Medline] [Order article via Infotrieve]
29. Marti HH, Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci U S A. 1998; 95: 15809–15814.
30. Tuder RM, Flook BE, Voelkel NF. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. Modulation of gene expression by nitric oxide. J Clin Invest. 1995; 95: 1798–1807.[Medline] [Order article via Infotrieve]
31. Waltenberger J, Mayr U, Pentz S, Hombach V. Functional upregulation of the vascular endothelial growth factor receptor KDR by hypoxia. Circulation. 1996; 94: 1647–1654.
32. Wang H, Olszewski B, Rosebury W, Wang D, Robertson A, Keiser JA. Impaired angiogenesis in SHR is associated with decreased KDR and MT1-MMP expression. Biochem Biophys Res Commun. 2004; 315: 363–368.[CrossRef][Medline] [Order article via Infotrieve]
33. Silvestre JS, Bergaya S, Tamarat R, Duriez M, Boulanger CM, Levy BI. Proangiogenic effect of angiotensin-converting enzyme inhibition is mediated by the bradykinin B(2) receptor pathway. Circ Res. 2001; 89: 678–683.
34. Silvestre JS, Kamsu-Kom N, Clergue M, Duriez M, Levy BI. Very-low-dose combination of the angiotensin-converting enzyme inhibitor perindopril and the diuretic indapamide induces an early and sustained increase in neovascularization in rat ischemic legs. J Pharmacol Exp Ther. 2002; 303: 1038–1043.
35. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004; 350: 2335–2342.
36. Mourad JJ, des Guetz G, Debbabi H, Levy BI. Blood pressure rise following angiogenesis inhibition by bevacizumab. A crucial role for microcirculation. Ann Oncol. 2008; 19: 927–934.
37. Henry TD, Rocha-Singh K, Isner JM, Kereiakes DJ, Giordano FJ, Simons M, Losordo DW, Hendel RC, Bonow RO, Eppler SM, Zioncheck TF, Holmgren EB, McCluskey ER. Intracoronary administration of recombinant human vascular endothelial growth factor to patients with coronary artery disease. Am Heart J. 2001; 142: 872– 880.[CrossRef][Medline] [Order article via Infotrieve]
38. Yang R, Ogasawara AK, Zioncheck TF, Ren Z, He GW, DeGuzman GG, Pelletier N, Shen BQ, Bunting S, Jin H. Exaggerated hypotensive effect of vascular endothelial growth factor in spontaneously hypertensive rats. Hypertension. 2002; 39: 815–820.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||