Remodeling Of Resistance Arteries Research Articles

LIM Kinase Inhibition Diminishes Hypertension and Vasoconstriction‐Induced Inward Remodeling in Mouse and Human Resistance Arteries

Vascular remodeling is an early biomarker of cardiovascular disease (CVD) related to structural changes in resistance arteries (RA). Arteriolar inward remodeling, characterized by a reduced luminal diameter that is associated with changes in the homeostasis of actin polymerization/depolymerization pathways, is posited to precede the development of hypertension. We hypothesize that inward remodeling involves changes in the actin cytoskeleton that facilitate cell motility and the rearrangement of vascular smooth muscle cells (VSMCs) around the vascular lumen. Moreover, we predict that inhibition of actin polymerization with a Lim kinase (LIMK) inhibitor would block the inward remodeling process associated with prolonged vasoconstriction and hypertension. We have previously reported in ex vivo experiments in rats that long‐term exposure of RA to vasoconstrictors induces vascular inward remodeling via activation of the Rho pathway, which in part promotes actin polymerization. We now demonstrate that VSMCs increase the inward remodeling ratio of filamentous to globular actin (F/G‐actin) in response to GPCR‐activation by a norepinephrine (NE) and angiotensin‐II (AngII). GPCR‐mediated Rho activates (LIMK), which subsequently phosphorylates and inactivates the actin severing enzyme, cofilin, thus effecting an increase in F/G‐actin ratios. In isolated rat cremasteric RA we have demonstrated that LIMK inhibition blocks the inward remodeling induced by of GPCR agonists and that this is coincident with a reduction in cofilin phosphorylation. To study the arterial remodeling process in vivo, we imaged the outer ear of a transgenic mouse line that expresses smooth muscle actin‐GFP in VSMCs by multiphoton microscopy. We treated these mice with AngII (1000ng/kg/min) for 14 days to induce vasoconstriction and hypertension. We imaged the same resistance artery within each ear before (Day 0) and after (Day 14) AngII infusion to study the progression of remodeling over time. Starting at day 0, one ear was injected every four days with a LIMK inhibitor (LIMKi), while the other ear received vehicle (control). We observed a significant inward remodeling in vessels of the control ear compared to those of the ear treated with LIMKi (p<0.05). We then assessed the effect of LIMKi in the inward remodeling process associated with ex vivo prolonged vasoconstriction in isolated human RA from omental tissues and studied by pressure myography under passive conditions before and after a 4h incubation in the presence of NE + AngII (control) or NE + AngII + LIMKi. Exposure to NE + AngII induced vascular inward remodeling that was completely ablated by presence of LIMKi and resulted in outward remodeling (p<0.05). These data indicate that LIMK plays a necessary role in the vasoconstriction‐hypertension vascular remodeling process that makes it a potential target for the treatment of CVDs associated with resistance artery inward remodeling.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vascular Smooth Muscle Remodeling in Conductive and Resistance Arteries in Hypertension.

Cardiovascular disease is a leading cause of death worldwide and accounts for >17.3 million deaths per year, with an estimated increase in incidence to 23.6 million by 2030. 1 Cardiovascular death represents 31% of all global deaths 2 -with stroke, heart attack, and ruptured aneurysms predominantly contributing to these high mortality rates. A key risk factor for cardiovascular disease is hypertension. Although treatment or reduction in hypertension can prevent the onset of cardiovascular events, existing therapies are only partially effective. A key pathological hallmark of hypertension is increased peripheral vascular resistance because of structural and functional changes in large (conductive) and small (resistance) arteries. In this review, we discuss the clinical implications of vascular remodeling, compare the differences between vascular smooth muscle cell remodeling in conductive and resistance arteries, discuss the genetic factors associated with vascular smooth muscle cell function in hypertensive patients, and provide a prospective assessment of current and future research and pharmacological targets for the treatment of hypertension.

Inositol 1,4,5-Trisphosphate Receptors in Hypertension.

Chronic hypertension remains a major cause of global mortality and morbidity. It is a complex disease that is the clinical manifestation of multiple genetic, environmental, nutritional, hormonal, and aging-related disorders. Evidence supports a role for vascular aging in the development of hypertension involving an impairment in endothelial function together with an alteration in vascular smooth muscle cells (VSMCs) calcium homeostasis leading to increased myogenic tone. Changes in free intracellular calcium levels ([Ca2+]i) are mediated either by the influx of Ca2+ from the extracellular space or release of Ca2+ from intracellular stores, mainly the sarcoplasmic reticulum (SR). The influx of extracellular Ca2+ occurs primarily through voltage-gated Ca2+ channels (VGCCs), store-operated Ca2+ channels (SOC), and Ca2+ release-activated channels (CRAC), whereas SR-Ca2+ release occurs through inositol trisphosphate receptor (IP3R) and ryanodine receptors (RyRs). IP3R-mediated SR-Ca2+ release, in the form of Ca2+ waves, not only contributes to VSMC contraction and regulates VGCC function but is also intimately involved in structural remodeling of resistance arteries in hypertension. This involves a phenotypic switch of VSMCs as well as an alteration of cytoplasmic Ca2+ signaling machinery, a phenomena tightly related to the aging process. Several lines of evidence implicate changes in expression/function levels of IP3R isoforms in the development of hypertension, VSMC phenotypic switch, and vascular aging. The present review discusses the current knowledge of these mechanisms in an integrative approach and further suggests potential new targets for hypertension management and treatment.

Clinical Implications

HomeHypertensionVol. 72, No. 1Clinical Implications Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBClinical Implications Originally published13 Jun 2018https://doi.org/10.1161/HYPERTENSIONAHA.118.11471Hypertension. 2018;72:2Blood Pressure Monitoring and Atrial Fibrillation (p 110)Download figureDownload PowerPointStrategies aiming at effective screening for atrial fibrillation (AF) are expected to contribute considerably in early AF detection and management and thereby efficient stroke prevention. Ambulatory blood pressure monitoring (ABPM) is currently proposed by several organizations as reference tool for confirming the diagnosis of hypertension and is increasingly used in clinical practice. A novel ABPM device with implemented algorithm for automated AF detection during each blood pressure measurement has been developed. This study assessed the diagnostic accuracy of this device for AF detection versus reference 24-hour Holter monitoring during routine 24-hour ABPM in elderly hypertensives. In ABPM reading-to-reading analysis, the diagnostic accuracy of this novel technology for AF detection seemed to be reasonable with high sensitivity and moderate specificity. A high frequency of AF false-positive ABPM readings was caused by excessive supraventricular ectopic activity, which, however, confers high risk for future AF. The receiver operating characteristics curve revealed that AF-positive readings at 26% during 24-hour ABPM had sensitivity 100% and specificity 85% for detecting paroxysmal AF (area under the curve, 0.91; P<0.01). These findings suggest that in elderly hypertensives a 24-hour ABPM recording with at least 26% of the readings suggesting AF indicates a high probability for AF diagnosis and should be regarded as an indication for performing 24-hour Holter monitoring.Endothelial PPARγ and Vascular Aging (p 227)Download figureDownload PowerPointAging is the greatest risk factor for vascular disease and its clinical complications, but insight into mechanisms that control vascular aging, particularly within individual cells, is very limited. Our ability to slow the progression of vascular aging is limited in part by our poor understanding of determinants of these changes. The transcription factor PPARγ (peroxisome proliferator–activated receptor-γ) has protective vascular effects when activated pharmacologically. To gain new insight into this area, we used mice expressing dominant negative PPARγ in endothelial cells (E-V290M) to test the hypothesis that interference with endothelial PPARγ augments age-induced vascular dysfunction. Because of their clinical importance in relation to stroke and cognitive deficits, we studied carotid arteries. Based on comparisons of E-V290M with nontransgenic littermates, our findings suggest age-related oxidative stress, inflammation, senescence, and endothelial dysfunction are increased after genetic interference with PPARγ, implicating an essential role for this molecule and cell type in protecting against key aspects of vascular aging. Loss of these beneficial effects because of disease, genetic changes, or protein alterations has implications for the rate of progression and severity of vascular aging, other forms of vascular disease, and potentially, effectiveness of therapeutic approaches. For example, dietary caloric restriction delays aging in many experimental models. Interestingly, PPARγ is one of the molecules with the strongest caloric restriction mimetic activity.Role of Endothelial BH4 in Vascular Remodeling (p 128)Download figureDownload PowerPointEvidence from experimental and clinical studies have demonstrated that reduced tetrahydrobiopterin (BH4) bioavailability, an essential cofactor for NO synthase, is associated with endothelial cell dysfunction and abdominal aortic aneurysm formation in response to Ang (Angiotensin) II. However, it is unknown if there is a specific requirement for endothelial cell BH4 in modulating susceptibility to structural vascular disease and if loss of endothelial cell BH4 impacts on pathological vascular remodeling. In this issue of Hypertension, we use an endothelial cell-specific knockout mouse (Gch1fl/flTie2cre mice) to investigate the requirement for endothelial cell BH4 in the cardiovascular response to Ang II. Even at a subpressor Ang II dose (0.4 mg/kg per day), we observed hypertension and pathological remodeling of both conduit and resistance arteries in Gch1fl/flTie2cre mice which were associated with eNOS (endothelial NO synthase) uncoupling and increased aortic eNOS-derived H2O2 generation. These findings indicate that selective endothelial cell BH4 deficiency is sufficient to cause pathophysiological changes in the vessel wall, in both large conduit vessels and small resistance vessels. Thus, targeting endothelial cell redox function, exemplified by Gch1 and BH4 biosynthesis, is not only a rational therapeutic target for improving endothelial functional but is also able to modify structural and pathological remodeling in cardiovascular disease. Previous Back to top Next FiguresReferencesRelatedDetails July 2018Vol 72, Issue 1 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.118.11471PMID: 30550717 Originally publishedJune 13, 2018 PDF download Advertisement

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

Changes in extracellular matrix in subcutaneous small resistance arteries of patients with essential hypertension

Background: In the development of hypertensive microvascular remodeling, a relevant role may be played by changes in extracellular matrix proteins. Aim of this study was the to evaluate some extracellular matrix components within the tunica media of subcutaneous small arteries in 9 normotensive subjects and 12 essential hypertensive patients, submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region.Patients and Methods: Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, fibronectin, laminin, transforming growth factor-beta-1 (TGF-β1) and emilin-1 contents within the tunica media were evaluated by immunofluorescence and relative immunomorphometrical analysis (immunopositivity % of area). The total collagen content and collagen subtypes within the tunica media were evaluated using both Sirius red staining (under polarized light) and immunofluorescence assay.Results: Normotensive controls had less total and type III collagen in respect with hypertensive patients. Fibronectin and TGF-β1 tunica media content was significantly greater in essential hypertensive patients, compared with normotensive controls, while laminin and emilin-1 tunica media content was lesser in essential hypertensive patients, compared with normotensive controls. A significant correlation was observed between fibronectin tunica media content and media to lumen ratio.Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, a relevant fibrosis may be detected; fibronectin and TGF-β1 tunica media content is increased, while laminin and emilin-1 content is decreased; these changes might be involved in the development of small resistance artery remodeling in humans.

Remodeling of Wall Mechanics and the Myogenic Mechanism of Rat Intramural Coronary Arterioles in Response to a Short-Term Daily Exercise Program: Role of Endothelial Factors

Purpose: Exercise elicits early adaptation of coronary vessels enabling the coronary circulation to respond adequately to higher flow demands. We hypothesized that short-term daily exercise induces biomechanical and functional remodeling of the coronary resistance arteries related to pressure. Methods: Male rats were subjected to a progressively increasing 4-week treadmill exercise program (over 60 min/day, 1 mph in the final step). In vitro pressure-diameter measurements were performed on coronary segments (119 ± 5 μm in diameter at 50 mm Hg) with microarteriography. The magnitude of the myogenic response and contribution of endogenous nitric oxide and prostanoid production to the wall mechanics and pressure-diameter relationship were assessed. Results: Arterioles isolated from exercised ani mals – compared to the sedentary group – had thicker walls, increased distensibility, and a decreased elastic modulus as a result of reduced wall stress in the low pressure range. The arterioles of exercised rats exhibited a more powerful myogenic response and less endogenous vasoconstrictor prostanoid modulation at higher pressures, while vasodilator nitric oxide modulation of diameter was augmented at low pressures (< 60 mm Hg). Conclusions: A short-term daily exercise program induces remodeling of rat intramural coronary arterioles, likely resulting in a greater range of coronary autoregulatory function (constrictor and dilator reserves) and more effective protection against great changes in intraluminal pressure, contributing thereby to the optimization of coronary blood flow during exercise.

Four-day pulse of sodium cromoglycate modulates pulmonary vessel wall remodeling during 21-day hypoxia in rats

ABSTRACTAim of the study: Remodeling of pulmonary resistance arteries in rats due to 4-day hypoxia could be successfully suppressed by sodium cromoglycate. In this study, we tested the difference in the suppression between two distinct time patterns of cromoglycate administration during 21-day hypoxia. In the experiment, we focused on some details in both smooth muscle cells and extracellular matrix of pulmonary arterial walls. Methods: During 21-day hypoxia, rats were treated with sodium cromoglycate either in the first four days or in the last four days. The first four days were chosen to test efficiency of an initial pulse of cromoglycate to suppress pulmonary vascular remodeling. The last four-day administration tested possibility to block remodeling post hoc. Results: Initial pulse reduced and modified remodeling in all levels of pulmonary arteries, which comprises neomuscularization of prealveolar arteries, asymmetrical hypertrophy of tunica media in muscular pulmonary arteries and hypertrophy of tunica media and tunica adventitia in large conduit arteries. Terminal pulse had only negligible effect. Conclusions: Only the initial cromoglycate therapy led to significant morphological suppression of remodeling. We therefore assume important role of initial remodeling influencing during long time hypoxia experiment.

The Role of Sex in the Pathophysiology of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterised by increased pulmonary vascular resistance and pulmonary artery remodelling as result of increased vascular tone and vascular cell proliferation, respectively. Eventually, this leads to right heart failure. Heritable PAH is caused by a mutation in the bone morphogenetic protein receptor-II (BMPR-II). Female susceptibility to PAH has been known for some time, and most recent figures show a female-to-male ratio of 4:1. Variations in the female sex hormone estrogen and estrogen metabolism modify FPAH risk, and penetrance of the disease in BMPR-II mutation carriers is increased in females. Several lines of evidence point towards estrogen being pathogenic in the pulmonary circulation, and thus increasing the risk of females developing PAH. Recent studies have also suggested that estrogen metabolism may be crucial in the development and progression of PAH with studies indicating that downstream metabolites such as 16α-hydroxyestrone are upregulated in several forms of experimental pulmonary hypertension (PH) and can cause pulmonary artery smooth muscle cell proliferation and subsequent vascular remodelling. Conversely, other estrogen metabolites such as 2-methoxyestradiol have been shown to be protective in the context of PAH. Estrogen may also upregulate the signalling pathways of other key mediators of PAH such as serotonin.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries

SummaryHypertension is a well‐established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age‐matched wild‐type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Microvascular structure as a prognostically relevant endpoint.

Remodelling of subcutaneous small resistance arteries, as indicated by an increased media-to-lumen ratio, is frequently present in hypertensive, obese, or diabetic patients. The increased media-to-lumen ratio may impair organ flow reserve. This may be important in the maintenance and, probably, also in the progressive worsening of hypertensive disease. The presence of structural alterations represents a prognostically relevant factor, in terms of development of target organ damage or cardiovascular events, thus allowing us a prediction of complications in hypertension. In fact, media-to-lumen ratio of small arteries at baseline, and possibly their changes during treatment may have a strong prognostic significance. However, new, non-invasive techniques are needed before suggesting extensive application of the evaluation of remodelling of small arteries for the cardiovascular risk stratification in hypertensive patients. Some new techniques for the evaluation of microvascular morphology in the retina, currently under clinical investigation, seem to represent a promising and interesting future perspective. The evaluation of microvascular structure is progressively moving from bench to bedside, and it could represent, in the near future, an evaluation to be performed in all hypertensive patients, to obtain a better stratification of cardiovascular risk, and, possibly, it might be considered as an intermediate endpoint in the evaluation of the effects of antihypertensive therapy, provided that a demonstration of a prognostic value of non-invasive measures of microvascular structure is made available.

Absence of Endothelial ERα Results in Arterial Remodeling and Decreased Stiffness in Western Diet-Fed Male Mice.

The role of estrogen receptor-α (ERα) signaling in the vasculature of females has been described under different experimental conditions and our group recently reported that lack of endothelial cell (EC) ERα in female mice fed a Western diet (WD) results in amelioration of vascular stiffness. Conversely, the role of ERα in the male vasculature in this setting has not been explored. In conditions of overnutrition and insulin resistance, augmented arterial stiffness, endothelial dysfunction, and arterial remodeling contribute to the development of cardiovascular disease. Here, we used a rodent model of decreased ERα expression in ECs [endothelial cell estrogen receptor-α knockout (EC-ERαKO)] to test the hypothesis that, similar to our findings in females, loss of ERα signaling in the endothelium of insulin-resistant males would result in decreased arterial stiffness. EC-ERαKO male mice and same-sex littermates were fed a WD (high in fructose and fat) for 20 weeks and then assessed for vascular function and stiffness. EC-ERαKO mice were heavier than littermates but exhibited decreased vascular stiffness without differences in endothelial-dependent vasodilatory responses. Mesenteric arteries from EC-ERαKO mice had significantly increased diameters, wall cross-sectional areas, and mean wall thicknesses, indicative of outward hypertrophic remodeling. This remodeling paralleled an increased vessel wall content of collagen and elastin, inhibition of matrix metalloproteinase activation and a decrease of the incremental modulus of elasticity. In addition, internal elastic lamina fenestrae were more abundant in the EC-ERαKO mice. In conclusion, loss of endothelial ERα reduces vascular stiffness in male mice fed a WD with an associated outward hypertrophic remodeling of resistance arteries.

Matrix Metalloproteinase (MMP)‐2 Diverges in Regulate Calponin‐1 in Conductance and Resistance Arteries in Early Hypertension‐Induced Vascular Dysfunction and Remodeling

IntroductionIncreased activity of MMP‐2 degrades many extra‐ and intracellular proteins, an effect that may contribute to hypertension‐induced vascular remodeling and dysfunction. Calponin‐1 is a differetiation marker of vascular smooth muscle cells (VSMC) and also inhibits Mg‐ATPase activity of myosin to regulate contraction. Calponin‐1 is an intracellular proteolytic target of MMP‐2 in aortas of hypertensive rats. The aim of this study is to investigate whether MMP‐2 contributes to hypertension‐induced vascular remodeling and dysfunction of conductance and resistance arteries by regulating calponin‐1.MethodsMale wistar rats were submitted to the two kidney‐one clip (2K‐1C) model of hypertension or sham surgery and were treated with doxycycline (inhibitor of MMPs, 30 mg/kg/day) or water for one week. Systolic blood pressure (SBP) was daily accessed by tail cuff plethysmography. After one week, aortas and mesenteric arteries were removed to analyze the activity of MMP‐2 by in situ zymography and calponin‐1 by immunofluorescence. Morphological analysis was performed with hematoxylin and eosin staining in aortas and pressurized myograph in mesenteric arteries. Immunohistochemistry to Ki‐67 was performed to analyze celular proliferation. Phenylephrine (PE) concentration‐effect curve was performed in aortas and in the third branch of mesenteric arteries by using tension myography. Statistical analysis were done by two‐way ANOVA. The Ethics Committee in Animal Research approved all protocols (012/2015‐1).ResultsSBP increased in the 2K‐1C rats (145.8±1.6 vs. 118.2±2.9, p&lt;0,05) and treatment with doxycycline did not reduce it. The morphological analysis showed that 2K‐1C aortas had an increase in the media per lumen ratio (p&lt;0,05) and cross‐sectional area (p&lt;0,05) vs. Sham groups, an alteration that was not observed in mesenteric arteries at early hypertension. Furthermore, doxycycline was able to revert the increased VSMCs proliferation in aortas of 2K‐1C rats (p&lt;0,05). On the other hand, PE significantly contracted more the mesenteric arteries of 2K‐1C (p&lt;0.05 vs. Sham), and doxycycline reverted it. The potency of PE to contract aortas of 2K‐1C rats was higher than controls and doxycycline also reverted it. MMP‐2 activity was increased in both arterial bed of 2K‐1C rats and doxycycline reduced it (p&lt;0,05). While calponin‐1 was lower in the aortas of 2K‐1C rats and treatment ameliorated it (p&lt;0,05), its levels were increased in the 2K‐1C mesenteric arteries (p&lt;0,05).ConclusionIn conductance arteries, the reduction of calponin‐1 by MMP‐2 contributed to VSMC proliferation and phenotype switch, which may result in the hypertrophic remodeling of hypertension. In resistance arteries, MMP‐2 may contribute to increase calponin‐1 as a regulatory mechanism to compensate hypertension‐induced increased vascular resistance. This effect may be particularly related to the extracellular mechanisms of MMP‐2.Support or Funding InformationCNPq, CAPES and FAPESP

Placental Stem Villus Arterial Remodeling Associated with Reduced Hydrogen Sulfide Synthesis Contributes to Human Fetal Growth Restriction

Intrauterine fetal growth restriction (IUGR) is often associated with compromised umbilical arterial flow, indicating increased placental vascular resistance. Oxidative stress is causatively implicated. Hydrogen sulfide maintains differentiated smooth muscle in vascular beds, and its synthetic enzymecystathionine-γ-lyase (CSE) is down-regulated in growth-restricted placentas. We hypothesized that remodeling of resistance arteries in stem villi contributes to IUGR by compromising umbilical blood flow via oxidative stress, reducing hydrogen sulfide signaling. Stem villus arteries in human IUGR placentasdisplaying absent or reversed end-diastolic flow contained reduced myosin heavy chain, smooth muscle actin, and desmin, and increased markers of dedifferentiation, cellular retinol-binding protein 1, and matrix metalloproteinase 2, compared to term and preterm controls. Wall thickness/lumen ratio was increased, lumen diameter decreased, but wall thickness remained unchanged in IUGR placentas. CSE correlated positively with myosin heavy chain, smooth muscle actin, and desmin. Birth weight correlated positively with CSE, myosin heavy chain, smooth muscle actin, and desmin, and negatively with cellular retinol-binding protein 1 and matrix metalloproteinase 2. These findings could be recapitulated invitro by subjecting stem villus artery explants to hypoxia-reoxygenation, or inhibiting CSE. Treatment with a hydrogen sulfide donor, diallyl trisulfide, prevented these changes. IUGR is associated with vascular remodeling of the stem villus arteries. Oxidative stress results in reduction of placental CSE activity, decreased hydrogen sulfide production, and smooth muscle cell dedifferentiation invitro. This vascular remodeling isreversible,and hydrogen sulfide donors are likely to improve pregnancy outcomes.

Estrogen deficiency modifies matrix metalloproteinases activity and vascular function of mesenteric arteries in female rats

Objective: Estrogen possesses vasoprotective effects and its deficiency has been implicated in the pathogenesis of postmenopausal hypertension. Reduced matrix metalloproteinases (MMPs) activity is accompanied with extracellular matrix (ECM) accumulation in large arteries, leading to the hypertensive arterial remodeling in resistance arteries. In the present study, we investigate whether estrogen deficiency induces alteration of MMP-mediated cleavage of vascular collagen in rats with ovariectomy (Ovx). Materials and Methods: Adult female rats were ovariectomized bilaterally to induce estrogen deficiency. Evolution of systolic blood pressure, diastolic blood pressure (DBP), and mean blood pressure (MBP) was monitored weekly until 12 weeks after Ovx in conscious rats. The vascular reactivity and time-course changes of collagen type I, MMP-2, membrane type 1-MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2) protein expression in mesenteric arteries were evaluated. Results: Compared with sham group, DBP and MBP significantly increased 5 weeks after Ovx, lasting to at least 12 weeks. Acetylcholine-induced vasodilatation of precontracted mesenteric rings significantly reduced 9 weeks after Ovx. Collagen type I accumulation in mesenteric arteries appeared 6 weeks after Ovx, which persisted till 12 weeks. The levels of latent and active MMP-2 did not show significant change until 12 weeks after Ovx. Moreover, MT1-MMP significantly downregulated during 1–4 weeks and soon recovered to normal levels. TIMP-2 reduced at 4 th week and gradually returned to normal levels during 6–12 weeks. Conclusion: Long-term estrogen deficiency results in a shift in ECM profiles and diminished MMPs activities, leading to remodeling of small arteries, which may be associated with postmenopausal hypertension. This study provides new insight into the pathophysiology of vascular remodeling in estrogen-deficient conditions.

Lentil consumption reduces resistance artery remodeling and restores arterial compliance in the spontaneously hypertensive rats

We previously established that lentils were able to significantly attenuate the development of hypertension in spontaneously hypertensive rats (SHRs), but the mechanism was not investigated. The current study was therefore designed to examine the effect of lentils on arterial function in relation to arterial stiffness, lipid biochemistry and activation of select aortic proteins. Seventeen-week-old male SHRs were randomly assigned to groups (n=10/group) fed (a) 30% w/w green lentils, (b) 30% red lentils, (c) 30% mixed lentils (red and green) or (d) no lentils for 8 weeks. Normotensive Wistar Kyoto (WKY) groups (n=10/group) received either the mixed lentil or no lentil diet. Blood pressure, pulse wave velocity and serum lipids were measured at baseline and 8 weeks, while pressure myography, arterial morphology and aortic proteins were measured after termination. There were no dietary-related changes in pulse wave velocity or blood pressure for any SHR or WKY group. Low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were significantly lower in only SHR red lentil and WKY mixed lentil groups compared to their controls. The lentil diets reduced the media:lumen ratio of SHRs relative to control-fed SHRs but had no effect on WKYs. Both red and green lentils reduced arterial stiffness of SHRs but not WKYs. SHR lentil groups showed lower aortic p38 mitogen-activated protein kinase (p38MAPK) phosphorylation, thus implying that p38MAPK activation is suppressed with lentil feeding. Lentil-based diets suppress pathological vascular remodeling in SHRs, while green lentils maintain the vascular function of SHRs similar to normotensive WKYs despite the presence of high blood pressure.

0259 : Neutrophil and thrombospondin-1 implications in flow-mediated remodeling of resistance arteries from mice

On vascular endothelium, shear stress produces vasorelaxation and outward arterial remodeling. A chronic increase in blood flow induces arterial diameter expansion (normalizing shear stress), and is associated with a compensatory hypertrophy to normalize wall strain. This remodeling is due to Inflammation and nitric oxide (NO) production in resistance arteries. A large number of studies have demonstrated that Thrombospondin-1 (TSP1), a homotrimeric glycoprotein, can have an inhibitory action on the NO pathway and a positive effect on inflammation in vitro . Nevertheless, the role of TSP1 on remodeling is poorly understood. We hypothesized that TSP1 is able to participate in this outward arterial remodeling and hypertrophy by immune cells recruitment. We investigated this remodeling in mesenteric resistance arteries of WT or TSP1-KO mice aged to 12-16 weeks were used. An IgG2a isotype control mAb 2A3 or Ly6G-specific mAb 1A8 are used for neutrophil depletion by in vivo administration. Ligation of mesenteric resistance arteries of WT/TSP1- KO and IgG2a/neutrophil-depleted mice allowed modifying blood flow in vivo, thus exposing arteries to low (LF), normal (NF), or high flow (HF). After 7 days, arteries were isolated for in vitro study. Phenylephrine-mediated contraction and Acetylcholine-mediated relaxation were stronger in HF than in NF vessels on WT and IgG2a control mice. Any difference between HF or NF in TSP1-KO and neutrophil-depleted mice were found. Furthermore, passive diameter analysis revealed a higher diameter in HF than NF in WT and IgG2a mice. These differences are lost in TSP1-KO and neutrophil-depleted mice. These results suggest that TSP1 are essential for remodeling in mesenteric arteries by promoting neutrophils recruitment. The author hereby declares no conflict of interest

Abstract P134: Identification of Tissue Inhibitor of Metalloproteinases (TIMP)-4 as a Novel PPARγ Target in Smooth Muscle Cell

Patients with PPARγ mutations develop severe early onset hypertension, type 2 diabetes and lipodystrophy. Our recent findings demonstrated that transgenic mice expressing a dominant negative (DN) PPARγ mutant in vascular smooth muscle (S-P467L) exhibited exacerbated DOCA-salt induced hypertension and vascular remodeling in conduit and resistance arteries. Here, we hypothesized that predisposition to vascular injury during hypertension in S-P467L mice is through altered expression of PPARγ target genes in smooth muscle cells (SMC). Gene expression profiling in aorta and mesenteric arteries revealed a significant loss of Tissue Inhibitor of Metalloproteinases (TIMP)-4 in S-P467L compared to non-transgenic (NT) littermates. Interference with PPARγ activity either by treatment with PPARγ inhibitor, GW9662 or expressing P467L PPARγ markedly suppressed TIMP-4 in primary SMC, suggesting that loss of TIMP-4 in S-P467L arteries is a direct result of PPARγ inhibition. Downregulation of TIMP-4 in SMC by GW9662 was correlated with a significant increase in total MMP activity (Fluorescent signal subtracted from negative control; vehicle: 1±0.5, GW9662: 2.2±0.7, p&lt;0.05), consistent with TIMP-4 function as an endogenous inhibitor of MMPs. Overexpressing TIMP-4 in SMC significantly blunted cell migration compared to those with empty plasmid (change in open area after 8 hr; Empty: 1±0.05, TIMP-4: 0.8±0.051, p&lt;0.05), whereas decreased TIMP-4 expression caused by mutant PPARγ resulted in increased migration (change in open area after 10 hr; GFP: 1±0.25, P467L: 1.63±0.26, p&lt;0.05). The significance of TIMP-4 was also underscored during hypertension since the compensatory increase in TIMP-4 during DOCA-salt in NT mesenteric arteries was lost in S-P467L. We identified two highly conserved potential PPAR response elements (PPREs) close to TIMP-4 promoter region using a sequence-based model. Chromatin immunoprecipitation assay showed strong binding of PPARγ at one of the suggested PPREs in SMC (% input±sem; GFP: 37±0.6, P467L: 66±0.7, p&lt;0.05 vs. IgG), suggesting that TIMP-4 is a direct target of PPARγ. Our findings highlight one of protective mechanisms of PPARγ during hypertension and provide a novel link between PPARγ and TIMP-4.

Abstract 024: The Direct Renin Inhibitor Aliskiren Improves Vasodilation and Endothelial Function in Resistance Arteries from Diabetic and Hypertensive Patients

We previously demonstrated that the direct renin inhibitor aliskiren (ALK) significantly reduced the remodeling of subcutaneous resistance arteries of hypertensive patients as compared to the angiotensin-converting enzyme inhibitor ramipril (RAM). Here we questioned whether endothelial function of resistance arteries would improve after 1 year of blood pressure (BP) control with ALK or RAM. Sixteen diabetic patients with mild essential hypertension were randomized to ALK (150-300 mg once daily, n=9) or RAM (5-10 mg once daily, n=7). Subcutaneous resistance arteries were dissected from gluteal biopsy and mounted on a pressurized micromyograph. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) and sodium nitroprusside (10 nM to 1 mM) respectively, in arteries pre-contracted with norepinephrine (10 μM). Carotid-femoral pulse wave velocity (PWV) was assessed by applantion tonometry. Forearm flow mediated dilation (FMD) was assessed by ultrasounds. The expression of P-eNOS/e-NOS and the markers of oxidative stress nitrotyrosine and LOX-1 were assessed by immunohistochemistry in resistance arteries. Patients were similar for age, sex, BMI and glycemic and metabolic control. Systolic BP was significantly and equally reduced by both ALK and RAM (153±8.9 mmHg reduced to 128±7.3 mmHg and 151±10.6 mmHg reduced to 121±12.1, respectively, P&lt;0.01) whereas diastolic BP was significantly reduced only in ALK-treated but not in RAM-treated patients (94.2±7.17 mmHg reduced to 81.4±6.31 mmHg, P&lt;0.01; and 84.7±12.22 reduced to 78.6±7.48, NS, respectively). PWV and FMD were similar in both groups before and after treatment. Endothelium-dependent vasodilation was improved only by ALK (max dilation 92.5±3.8% vs 50.5±14%, P&lt;0.05) but not by RAM (max dilation 72.9±5.6% vs 61.8±14.5%, NS). Endothelium-independent vasodilation was similar in all the groups. Only ALK increased P-eNOS/eNOS expression (+44% vs before treatment, p&lt;0.05). The markers of oxidative stress were similar in both groups before and after treatment. In conclusion ALK improved endothelial function and induced vasodilation in resistance arteries from diabetic and hypertensive patients.

Abstract P252: Effect of Maternal Diabetes on Fetal Programming of Vascular Remodeling Mechanisms in Adult Rats

Modifications of the intra-uterine environment are now recognized as an important cause of fetal stress. Leading to several responses such as loss of structure/function and pre-emptive adaptations to an adverse post-natal environment, and finally to adult diseases. It has been demonstrated that children of diabetic mothers have an increased risk of developing cardiovascular diseases (i.e. hypertension) during adulthood. Blood vessels are able to reorganize their structure in response to physiological alteration of blood flow or pathological stimuli. Constrictive vascular remodeling is currently associated with the occurrence of cardiovascular diseases. Thus, our objective was to study vascular remodeling in case of in utero exposure to maternal diabetes. This is in order to investigate the effect of intra-uterine environment perturbations on vascular fetal programming. We have developed an animal model of rats exposed in utero to moderated maternal hyperglycemia (DMO), which developed an hypertension around 6 months of age. We analyzed structure of elastic and resistance arteries (internal diameter, MCSA, intima-media thickness and remodeling parameters) in absence or presence of established hypertension in male DMO compared to controls (CMO) (at 3 and 18 months of age). Moreover, by in vivo sequential ligation of mesenteric arteries, we have studied vascular remodeling of resistance arteries in response to low (LF), normal (NF) or high flow (HF). In old DMO, we did not observe any vascular remodeling induced by hypertension neither in aorta nor in mesenteric arteries. This could be related to the increase of smooth muscle cell attachments that we observed. Moreover, after 1 or 3 weeks of mesenteric arteries ligation, DMO did not exhibit any constrictive remodeling in LF although expansive remodeling in HF is maintained. Interestingly, we measured an increase of eNOS activity and GP91 in LF arteries of theses animals although transglutaminase-2 protein expression was not modified. Our results demonstrate that in utero exposure to maternal diabetes induce modification of vascular remodeling mechanisms in adulthood. Absence of vascular response could be a pre-emptive adaptation to fetal programmed-hypertension in these animals.