Stephanie Lehoux

ECM remodeling in hypertensive heart disease

Hypertensive heart disease (HHD) occurs in patients that clinically have both diastolic and systolic heart failure and will soon become the most common cause of heart failure. Two key aspects of heart failure secondary to HHD are the relatively highly prevalent LV hypertrophy and cardiac fibrosis, caused by changes in the local and systemic neurohormonal environment. The fibrotic state is marked by changes in the balance between MMPs and their inhibitors, which alter the composition of the ECM. Importantly, the fibrotic ECM impairs cardiomyocyte function. Recent research suggests that therapies targeting the expression, synthesis, or activation of the enzymes responsible for ECM homeostasis might represent novel opportunities to modify the natural progression of HHD.

Molecular mechanisms of the vascular responses to haemodynamic forces.

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. Significant variations in mechanical forces, of physiological or physiopathological nature, occur in vivo. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, producing structural modifications of the arterial wall. In all the cases, vascular remodelling can be allotted to a modification of the tensional strain or shear, and underlie a trend to reestablish baseline mechanical conditions. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Mechanical forces also initiate complex signal transduction cascades, including nuclear factor‐κB and mitogen‐activated protein kinase pathways, leading to functional changes within the cell.

Signal Transduction of Mechanical Stresses in the Vascular Wall

The vascular wall is constantly subjected to a variety of mechanical forces in the form of stretch (tensile stress), due to blood pressure, and shear stress, due to blood flow. Alterations in either of these stresses are known to result in vascular remodeling, an adaptation characterized by modified morphology and function of the blood vessels, allowing the vessels to cope with physiological or pathological conditions. The processes involved in vascular remodeling include cellular hypertrophy and hyperplasia, as well as enhanced protein synthesis or extracellular matrix protein reorganization. In vitro studies using vascular cells have attempted to identify the mechanisms behind structural alterations. Possible pathways include ion channels, integrin interaction between cells and the extracellular matrix, activation of various tyrosine kinases (such as c-Src, focal adhesion kinase, and mitogen-activated protein kinases), and autocrine production and release of growth factors. These pathways lie upstream of de novo synthesis of immediate response genes and total protein synthesis, both of which are likely to be involved in the process of vascular remodeling.

Cellular mechanics and gene expression in blood vessels

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow, and shear stress. Alterations in stretch or shear stress invariably produce transformations in the vessel wall that will aim to accommodate the new conditions and to ultimately restore basal levels of tensile stress and shear stress. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Beyond the structural modifications incurred, mechanical forces can also initiate complex signal transduction cascades leading to functional changes within the cell. Many intracellular pathways, including the MAP kinase cascade, are activated by flow or stretch and initiate, via sequential phosphorylations, the activation of transcription factors and subsequent gene expression.

p47phox-Dependent NADPH Oxidase Regulates Flow-Induced Vascular Remodeling

Chronic alterations in blood flow elicit an adaptive response that tends to normalize shear stress, involving nitric oxide (NO) and matrix metalloproteinases (MMPs). To evaluate the role of NADPH oxidase in this process, we developed a new model of mouse arteriovenous fistula (AVF) connecting the right common carotid artery (RCCA) with the jugular vein, which does not affect blood pressure. Mice deficient for gp91phox and p47phox subunits of NADPH and wild-type controls were used. AVF greatly increased RCCA blood flow (0.78±0.12 to 4.71±0.78 mL/min; P<0.01), producing an abrupt rise in shear stress (35±1 to 261±17 dynes/cm2; P<0.01) within 24 hours. RCCA diameter (460±14 &mgr;m) gradually enlarged 1 and 3 weeks after AVF (534±14 &mgr;m and 627±19 &mgr;m; P<0.01), reducing shear stress (173±13 and 106±10 dynes/cm2, respectively). In gp91phox−/− mice, changes in RCCA caliber and shear stress matched controls. However, p47phox−/− mouse RCCAs enlarged only marginally, such that shear stress remained high (199±8 dynes/cm2 at 3 weeks). Likewise, remodeling was minimal in endothelial NO synthase (eNOS)−/− mice. In both control and gp91phox−/− animals, reactive oxygen species (ROS) production and MMP induction was enhanced by AVF, whereas in p47phox−/− and eNOS−/− mice such response was negligible. Similarly, nitrotyrosine staining, indicating peroxynitrite formation, was more pronounced in control and gp91phox−/− mice than in p47phox−/− and eNOS−/− mice. Hence, shear stress induces vascular NADPH oxidase comprising p47phox but not gp91phox. Generated ROS interact with NO to produce peroxynitrite, which in turn activates MMPs, facilitating vessel remodeling. Our study provides the first evidence that ROS play a fundamental role in flow-induced vascular enlargement.

Chronic Physiological Shear Stress Inhibits Tumor Necrosis Factor–Induced Proinflammatory Responses in Rabbit Aorta Perfused Ex Vivo

Background—Regions in the vasculature exposed to steady laminar flow have a lower likelihood for atherosclerosis than regions exposed to disturbed flow with low shear stress. We previously found that laminar flow of short duration inhibited tumor necrosis factor (TNF)-&agr;–mediated proinflammatory signaling in cultured endothelial cells (ECs). However, mechanisms responsible for the atheroprotective effects of physiological shear stress remain undefined. Therefore, we examined the effects of chronic shear stress on TNF-&agr;–induced inflammatory responses using an ex vivo perfusion organ culture system. Methods and Results—Rabbit aortas were exposed to low or normal shear stress (0.4 or 12 dyne/cm2) at a constant pressure for 24 to 26 hours. EC and vascular smooth muscle cell (VSMC) proteins were selectively purified. After exposure to low shear stress, TNF-&agr; (50 ng/mL, 6 hours) specifically stimulated vascular cell adhesion molecule (VCAM)-1 expression in ECs but not VSMCs. TNF-&agr;–stimulated VCAM expression was inhibited significantly by preexposure to normal shear stress. Normal shear stress inhibited TNF (15 minutes) activation of mitogen-activated protein (MAP) kinases (c-Jun NH2-terminal kinase [JNK], p38, extracellular signal–regulated kinase [ERK]) in ECs. Specific pharmacological inhibitors of JNK and p38 but not ERK significantly inhibited TNF-induced VCAM expression. Normal shear stress prevented the association of TNF receptor (TNFR)-1 with TNFR-associated factor (TRAF)-2. There was no effect of low or normal shear stress on TNF-&agr;–induced nuclear factor-&kgr;B activation. A nitric oxide synthesis inhibitor, NG-nitro-l-arginine methyl ester, did not reverse the inhibitory effects of shear stress on VCAM expression. Conclusions—These results suggest that physiological shear stress is antiinflammatory by specifically inhibiting MAP kinase signaling and inhibiting TRAF-2 interaction with TNFR-1.

Microparticles From Human Atherosclerotic Plaques Promote Endothelial ICAM-1–Dependent Monocyte Adhesion and Transendothelial Migration

Rationale and Objective: Membrane-shed submicron microparticles (MPs) released following cell activation or apoptosis accumulate in atherosclerotic plaques, where they stimulate endothelial proliferation and neovessel formation. The aim of the study was to assess whether or not MPs isolated from human atherosclerotic plaques contribute to increased endothelial adhesion molecules expression and monocyte recruitment. Method and Results: Human umbilical vein and coronary artery endothelial cells were exposed to MPs isolated from endarterectomy specimens (n=62) and characterized by externalized phosphatidylserine. Endothelial exposure to plaque, but not circulating, MPs increased ICAM-1 levels in a concentration-dependant manner (3.4-fold increase) without affecting ICAM-1 mRNA levels. Plaque MPs harbored ICAM-1 and transferred this adhesion molecule to endothelial cell membrane in a phosphatidylserine-dependent manner. MP-borne ICAM-1 was functionally integrated into cell membrane as demonstrated by the increased ERK1/2 phosphorylation following ICAM-1 ligation. Plaque MPs stimulated endothelial monocyte adhesion both in culture and in isolated perfused mouse carotid. This effect was also observed under flow condition and was prevented by anti–LFA-1 and anti–ICAM-1 neutralizing antibodies. MPs isolated from symptomatic plaques were more potent in stimulating monocyte adhesion than MPs from asymptomatic patients. Plaque MPs did not affect the release of interleukin-6, interleukin-8, or MCP-1, nor the expression of VCAM-1 and E-selectin. Conclusion: These results demonstrate that MPs isolated from human atherosclerotic plaques transfer ICAM-1 to endothelial cells to recruit inflammatory cells and suggest that plaque MPs promote atherosclerotic plaque progression.

Role of Matrix Metalloproteinases in Early Hypertensive Vascular Remodeling

Hypertension is associated with vascular remodeling characterized by rearrangement of extracellular matrix proteins. To evaluate how matrix metalloproteinase (MMP)-9 contributes to the progression of hypertensive vascular disease in vivo, wild-type (wt) or MMP-9−/− mice were treated with angiotensin II (Ang II; 1 &mgr;g/kg per minute, by minipump) plus a 5% NaCl diet during 10 days. Baseline blood pressure was equivalent in wt and knockout mice, but Ang II treatment increased systolic blood pressure to a greater extent (P<0.05) in MMP-9−/− mice (94±6 to 134±6 mm Hg; P<0.001) than in wt animals (93±4 to 114±6 mm Hg; P<0.01). In wt mice, Ang II treatment increased the carotid artery pressure–diameter relationship significantly, and maximal diameter reached 981±19 &mgr;m (P<0.01 versus sham; 891±10 &mgr;m). In contrast, in MMP-9−/− mice, carotid artery compliance was actually reduced after Ang II (P<0.05), and maximal diameter only reached 878±13 &mgr;m. Ang II treatment induced MMP-2 and increased carotid media thickness equally in both phenotypes. However, MMP-9 induction and in situ gelatinase activity were only enhanced in Ang II-treated wt mice, and vessels from these mice also produced more collagen I breakdown products than their MMP-9−/− counterparts (P<0.05). Inversely, staining for collagen IV was particularly enhanced in vessels from MMP-9−/− mice treated with Ang II. These results demonstrate the following: (1) the onset of Ang II–induced hypertension is accompanied by increased MMP-9 activity in conductance vessels; (2) absence of MMP-9 activity results in vessel stiffness and increased pulse pressure; and (3) MMP-9 activation is associated with a beneficial role early on in hypertension by preserving vessel compliance and alleviating blood pressure increase.

Extracellular matrix alterations in hypertensive vascular remodeling.

Vascular cells are very sensitive to their hemodynamic environment. Any change in blood pressure or blood flow can be sensed by endothelial and vascular smooth muscle cells and ultimately results in structural modifications within the vascular wall that accommodate the new conditions. In the case of hypertension, the increase in arterial stretch stimulates vessel thickening to normalize the tensile forces. This process requires modification of the extracellular matrix and of cell-matrix interactions, which mainly involves extracellular proteases. In hypertension, chronic exposure of the arterial wall to stretch leads to vascular remodeling, arterial stiffness and calcification, which finally affect target organ function. This review surveys how mechanical stretch regulates extracellular proteases, considering the signaling pathways involved and the consequences on the cardiovascular system.

Differential Regulation of Vascular Focal Adhesion Kinase by Steady Stretch and Pulsatility

Background—In vivo tensile strain in arteries comprises 2 components: steady stretch and pulsatile stretch. However, little attention has been paid to the differential transduction of these stimuli in whole vessels. Methods and Results—Using rabbit aortas maintained in organ culture for 24 hours, we found that focal adhesion kinase (FAK) was strongly activated by high intraluminal pressure (150 mm Hg), as evidenced by increased phosphorylation (P<0.01) of tyrosine residues Tyr-397 (3.06±0.17-fold), Tyr-407 (3.71±0.65-fold), Tyr-861 (1.92±0.33-fold), and Tyr-925 (2.41±0.39-fold), compared with 80 mm Hg controls. Immunohistochemistry showed positive staining for these phosphotyrosines in the endothelium and innermost smooth muscle cell layers. Total FAK phosphorylation was reduced in vessels at 150 mm Hg by treatment with the Src family kinase inhibitor PP2 or with the integrin–extracellular matrix interaction–blocking RGD peptide, attaining 1.75±0.22-fold and 2.00±0.19-fold, respectively (P<0.05), compared with 3.07±0.38-fold (P<0.001) in untreated vessels. PP2 prevented phosphorylation of Tyr-407 and Tyr-925, whereas RGD peptide abolished phosphorylation of Tyr-397 and Tyr-407. PP2 and RGD peptide also inhibited high pressure–induced binding of FAK with the effector Grb2 and blocked activation of extracellular regulated kinase (ERK) 1/2 in vessels at 150 mm Hg. In contrast, 10% cyclic stretch in aortas did not induce significant FAK phosphorylation relative to nonpulsatile controls. Furthermore, although ERK1/2 was activated in vessels exposed to pulsatility, it was not blocked by PP2 or RGD peptide treatment. Conclusions—Our results demonstrate that (1) steady and cyclic modes of stretch are transduced differently in the aorta, the former implicating FAK, the latter not, and (2) Src and integrins are involved in steady pressure–induced FAK.