THE IMPACT OF OUTWARD REMODELING ON VASODILATION IN SKELETAL MUSCLE RESISTANCE ARTERIES

Peripheral arterial occlusive disease (PAOD) is an ischemic disease characterized by narrowing of the peripheral arteries due to the accumulation of atherosclerotic plaque in the inner lining of the vessels, which disrupts blood flow to downstream tissues. Blood can be redirected into collateral vessels, natural bypasses around arterial occlusions, causing shear-induced outward remodeling of the vessels. The enlarged vessels facilitate transfer of increased blood flow to downstream tissues. The remodeling process, however, may impair vasodilation, which in turn may cause or contribute to intermittent claudication- transient pain brought on by locomotion. To stimulate the growth of collateral arteries, the femoral arteries of young, otherwise healthy mice were ligated distally to the profunda femoris, the stem to the gracilis collateral circuit. The diameter of the profunda femoris artery was measured at rest and following gracilis muscle contraction 7 and 28 days post-surgery using intravital microscopy. Enlarged resting diameter, consistent with collateral enlargement, and impaired vasodilation was observed at day 7, but not at day 28. To determine if impaired functional vasodilation is due to impaired endothelial- or smooth muscle-dependent responses during outward remodeling, cell-dependent vasodilators were applied to the hindlimb. Endothelial- and smooth muscle-dependent vasodilation was significantly impaired 7 days post-ligation, but not 28 days after. This data supports the hypothesis that smooth muscle dysfunction causes impaired functional vasodilation in the early stages of collateral enlargement

Piezo1: Proteins for mechanotransduction and integration of endothelial shear stress & intravascular pressure

Piezo proteins are transmembrane ion channels, specialized in detecting mechanosensitive stimuli and transduce mechanical forces into biochemical signals. Piezo proteins research has helped understand physiological mechanisms, but the integrative role that Piezo1 plays in the regulation of the microvasculature has remained unstudied. Our main objective was to characterize ex vivo microvascular responses to the blockade of Piezo1 mechanotransduction in male (n=29) and female (n=24) Sprague-Dawley (SD) rats. Gracilis arterioles (GA) and middle cerebral arterioles (MCA) were harvested for ex-vivo vessel preparations. After vessel viability confirmation, every vessel was submitted to myogenic and flow challenges under control conditions and after Grammostola Mechanotoxin 4 (GsMTx4) incubation to blocking Piezo1 channels, to quantify the homeostatic response of arterioles before and after Piezo1 antagonism. We are able to report Piezo1 as indispensable component in vascular smooth muscle cells (VSMC) and Endothelial cells (EC) to sense and change vessel diameter based on intravascular pressure and shear stress, correspondingly. Also, we report for the first time a heterogeneous response in males and females after Piezo1 antagonism in representative resistance arterioles from the skeletal muscle and cerebral circulation

Functional and structural modifications associated with hypertension, obesity and diabetes in the resistance vasculature

"December 2014."Dissertation Supervisors: Dr. Luis A. Martinez-Lemus and Luis Polo-Parada.Includes vita.Cardiovascular diseases are considered the leading cause of death nowadays. Hypertension, obesity and type-2 diabetes are deemed major risk factors for the development of cardiovascular diseases. In essential hypertension, one of the most important structural changes is the inward remodeling of the resistance arteries. I found that the mechanical properties of inwardly remodeled cremasteric-arterioles from rats are affected. Furthermore, it is the F-actin components of the cytoskeleton the ones that are strongly modified. In old spontaneously hypertensive rats, my results showed that, resistance arteries undergo hypertrophic inward remodeling; and, adrenergicvasoconstriction and vasodilation pathways are impaired. In diet-induced-obesity, micemesenteric arterioles were observed to undergo remodeling of the extracellular matrix components. Obesity and type-2 diabetes have been associated with insulin resistance, endothelial dysfunction and arterial stiffening. Jejunal-submucosal arterioles from diabetic obese patients had a reduced vasorelaxation to insulin in comparison to obese non-diabetics, while acetylcholine-vasodilation was similar in both groups. Reduced amounts of the subunit-alpha of the insulin receptor and MMP-9 were found in diabetics as well. This suggests that, in type-2 diabetes, the presence of a blunted insulinvasodilation response is a form of endothelial dysfunction that is not correlated with the body-to-mass index, but whose mechanism may be related with the activity of MMPs.Includes bibliographical references (pages 124-146)

Vascular Reactivity in Newly-Formed and Mature Arterialized Collateral Capillaries

Peripheral arterial occlusive disease (PAOD) is a globally-prevalent cardiovascular disease in which atherosclerotic plaques narrow arterial lumen diameters and restrict blood flow to downstream tissues. The impact of these occlusions can be mitigated by collateral vessels that connect parallel arterial branches and act as natural bypasses to maintain perfusion. In animal models that lack collateral arterioles, capillaries that connect terminal arteriolar segments can arterialize and form functional collaterals following an ischemic event; however, in the early stages of development, vasodilation is impaired. We explored the mechanism of impaired vasodilation in arterialized collateral capillaries (ACCs) and pre-existing collaterals (PECs) by evaluating endothelial-dependent vasodilation and endothelial-independent reactivity at day seven following the ischemic event. We also evaluated functional vasodilation in mature ACCs and PECs at day 21 by applying vasodilation inhibitors during the electrical stimulation of muscle contraction. Arterial occlusion was performed by ligating the cranial-lateral spinotrapezius feed artery in Balb/C mice, a strain that either lacks native arteriolar collaterals or contains a single collateral arteriole (~50% of mice), as opposed to the C57Bl/6 strain, which each contain 10 or more collateral arterioles. At seven days post-surgery, both vasodilation and vasoconstriction were impaired in ACCs when compared to terminal arterioles of similar size in unoperated limbs, but still exhibited significant changes when compared to baseline. The comparable reactivity in both endothelial-dependent and independent vasodilation at day-seven in ACCs indicates that vascular smooth muscle cells are likely responsible for the impairment, as they may still be developing, rearranging, or both, and are not yet fully capable of regulating diameter in immature ACCs. However, by 21 days post-ligation, ACCs regained the capacity to dilate in response to muscle contraction, and utilized similar vasodilation pathways as control vessels. At seven days post-ligation, PECs had impaired endothelialindependent dilation, but successful endothelial-dependent dilation, indicating the use of alternative pathways to dilate. Unlike ACCs, the PECs never completely restored vasodilation capabilities by day 21, which may be due to a variation in smooth muscle phenotype, sensitivity to vasoactive agents, and/or limited growth factor expression. For future work, evaluating collateral formation and vasodilation in a diseased model and investigating molecular variations in the smooth muscle may yield additional knowledge that can improve therapies for patients during ischemic events

Vascular smooth muscle contraction in hypertension

Hypertension is a major risk factor for many common chronic diseases, such as heart failure, myocardial infarction, stroke, vascular dementia and chronic kidney disease. Pathophysiological mechanisms contributing to the development of hypertension include increased vascular resistance, determined in large part by reduced vascular diameter due to increased vascular contraction and arterial remodelling. These processes are regulated by complex interacting systems such as the renin angiotensin aldosterone system (RAAS), sympathetic nervous system, immune activation and oxidative stress, which influence vascular smooth muscle function. Vascular smooth muscle cells are highly plastic and in pathological conditions undergo phenotypic changes from a contractile to a proliferative state. Vascular smooth muscle contraction is triggered by an increase in intracellular free calcium concentration ([Ca2+]i), promoting actin-myosin cross-bridge formation. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species and reorganization of the actin cytoskeleton. Activation of immune/inflammatory pathways and noncoding RNAs are also emerging as important regulators of vascular function. Vascular smooth muscle cell [Ca2+]i, not only determines the contractile state but also influences activity of many calcium-dependent transcription factors and proteins thereby impacting the cellular phenotype and function. Perturbations in vascular smooth muscle cell signaling and altered function influence vascular reactivity and tone, important determinants of vascular resistance and blood pressure. Here we discuss mechanisms regulating vascular reactivity and contraction in physiological and pathophysiological conditions and highlight some new advances in the field, focusing specifically on hypertension

Vascular Smooth Muscle as a Therapeutic Target in Disease Pathology

Our circulatory system is composed of numerous elements that are responsible for transport of blood and delivery of essential nutrients and gases to vital downstream tissues. Among these components that make up our circulation is vascular smooth muscle (VSM), the primary muscular and contractile element of blood vessels and regulator of many blood vessel functions. This is of particular importance as cardiovascular disease (CVD), the number one killer of individuals in America and worldwide, is primarily vascular in origin. Logically, identifying and characterizing feasible targets that could control CVD are highly appealing and much desired. With this in mind and given its centrality in control of vascular physiology, VSM has gained wide attention as a plausible target to combat elements of CVD. This book chapter focuses on VSM as a potential therapeutic target against CVD and will provide overview of vascular anatomy and physiology and brief discussions about the pivotal roles of VSM in CVD pathology, the influence of abnormal blood flow mechanics and hemodynamics in CVD, neural control of VSM and the vasculature, and possible novel cellular and molecular signaling targets that could be used to control and/or minimize CVD. This chapter hopes to serve as a valuable resource for basic and applied scientists as well as clinicians interested in understanding the crucial roles that VSM plays in vessel physiology and pathology

Age-related endothelial dysfunction in human skeletal muscle feed arteries: the role of free radicals derived from mitochondria in the vasculature

Aim This study sought to determine the role of free radicals derived from mitochondria in the vasculature in the recognized age-related endothelial dysfunction of human skeletal muscle feed arteries (SMFAs). Methods A total of 44 SMFAs were studied with and without acute exposure to the mitochondria-targeted antioxidant MitoQ and nitric oxide synthase (NOS) blockade. The relative abundance of proteins from the electron transport chain, phosphorylated (p-) to endothelial (e) NOS ratio, manganese superoxide dismutase (MnSOD) and the mitochondria-derived superoxide () levels were assessed in SMFA. Endothelium-dependent and endothelium-independent SMFA vasodilation was assessed in response to flow-induced shear stress, acetylcholine (ACh) and sodium nitroprusside (SNP). Results MitoQ restored endothelium-dependent vasodilation in the old to that of the young when stimulated by both flow (young: 68 ± 5; old: 25 ± 7; old + MitoQ 65 ± 9%) and ACh (young: 97 ± 4; old: 59 ± 10; old + MitoQ: 98 ± 5%), but did not alter the initially uncompromised, endothelium-independent vasodilation (SNP). Compared to the young, MitoQ in the old diminished the initially elevated mitochondria-derived levels and appeared to attenuate the breakdown of MnSOD. Furthermore, MitoQ increased the ratio of p-eNOS to NOS and the restoration of endothelium-dependent vasodilation in the old by MitoQ was ablated by NOS blockade. Conclusion This study demonstrated that MitoQ reverses age-related vascular dysfunction by what appears to be an NO-dependent mechanism in human SMFAs. These findings suggest that mitochondria-targeted antioxidants may have utility in terms of counteracting the attenuated blood flow and vascular dysfunction associated with advancing age

Vascular smooth muscle cells and arterial stiffening : relevance in development, aging, and disease

The cushioning function of large arteries encompasses distension during systole and recoil during diastole which transforms pulsatile flow into a steady flow in the microcirculation. Arterial stiffness, the inverse of distensibility, has been implicated in various etiologies of chronic common and monogenic cardiovascular diseases and is a major cause of morbidity and mortality globally. The first components that contribute to arterial stiffening are extracellular matrix (ECM) proteins that support the mechanical load, while the second important components are vascular smooth muscle cells (VSMCs), which not only regulate actomyosin interactions for contraction but mediate also mechanotransduction in cell-ECM homeostasis. Eventually, VSMC plasticity and signaling in both conductance and resistance arteries are highly relevant to the physiology of normal and early vascular aging. This review summarizes current concepts of central pressure and tensile pulsatile circumferential stress as key mechanical determinants of arterial wall remodeling, cell-ECM interactions depending mainly on the architecture of cytoskeletal proteins and focal adhesion, the large/small arteries cross-talk that gives rise to target organ damage, and inflammatory pathways leading to calcification or atherosclerosis. We further speculate on the contribution of cellular stiffness along the arterial tree to vascular wall stiffness. In addition, this review provides the latest advances in the identification of gene variants affecting arterial stiffening. Now that important hemodynamic and molecular mechanisms of arterial stiffness have been elucidated, and the complex interplay between ECM, cells, and sensors identified, further research should study their potential to halt or to reverse the development of arterial stiffness

Endothelial Mechanosignaling: Does One Sensor Fit All?

Significance: Forces are important in the cardiovascular system, acting as regulators of vascular physiology and pathology. Residing at the blood vessel interface, cells (endothelial cell, EC) are constantly exposed to vascular forces, including shear stress. Shear stress is the frictional force exerted by blood flow, and its patterns differ based on vessel geometry and type. These patterns range from uniform laminar flow to nonuniform disturbed flow. Although ECs sense and differentially respond to flow patterns unique to their microenvironment, the mechanisms underlying endothelial mechanosensing remain incompletely understood. Recent Advances: A large body of work suggests that ECs possess many mechanosensors that decorate their apical, junctional, and basal surfaces. These potential mechanosensors sense blood flow, translating physical force into biochemical signaling events. Critical Issues: Understanding the mechanisms by which proposed mechanosensors sense and respond to shear stress requires an integrative approach. It is also critical to understand the role of these mechanosensors not only during embryonic development but also in the different vascular beds in the adult. Possible cross talk and integration of mechanosensing via the various mechanosensors remain a challenge. Future Directions: Determination of the hierarchy of endothelial mechanosensors is critical for future work, as is determination of the extent to which mechanosensors work together to achieve force-dependent signaling. The role and primary sensors of shear stress during development also remain an open question. Finally, integrative approaches must be used to determine absolute mechanosensory function of potential mechanosensors. Antioxid. Redox Signal. 25, 373–388