Objective-Despite the role that extracellular matrix (ECM) plays in vascular signaling, little is known of the complex structural arrangement between specific ECM proteins and vascular smooth muscle cells. Our objective was to examine the hypothesis that adventitial elastin fibers are dominant in vessels subject to longitudinal stretch. Methods and Results-Cremaster muscle arterioles were isolated, allowed to develop spontaneous tone, and compared with small cerebral arteries. 3D confocal microscopy was used to visualize ECM within the vessel wall. Pressurized arterioles were fixed and stained with Alexa 633 hydrazide (as a nonselective ECM marker), anti-elastin, or anti-type 1 collagen antibody and a fluorescent nuclear stain. Exposure of cremaster muscle arterioles to elastase for 5 minutes caused an irreversible lengthening of the vessel segment that was not observed in cerebral arteries. Longitudinal elastin fibers were demonstrated on cremaster muscle arterioles using 3D imaging but were confirmed to be absent in cerebral vessels. The fibers were also distinct from type I collagen fibers and were degraded by elastase treatment. Key Words: cell physiology Ⅲ extracellular matrix Ⅲ microcirculation Ⅲ vascular biology Ⅲ elastin T he extracellular matrix (ECM) contains a number of proteins, including collagen, elastin, laminin, fibronectin, vitronectin, glycoproteins, and proteoglycans. In addition to providing a mechanically dynamic structural scaffold, the ECM is involved in physiological processes such as cell growth, differentiation, and migration. With respect to the vasculature, recent studies have demonstrated a number of these ECM proteins to actively signal through outside-in means into both vascular smooth muscle and endothelial cells, particularly conveying mechanical signals. For example, fibronectin binding through cell surface integrins modulates the activity of smooth muscle cell (SMC) ion channels (voltage-gated Ca 2ϩ channels and large conductance Ca 2ϩ -activated K ϩ channels) 1-3 and affects local cellular contractions. 4 Similarly, ECM protein-integrin activation of various intracellular signaling mechanisms underlies endothelial cell mechanotransduction to stimuli such as shear stress. 5,6 Despite these demonstrated roles of the ECM in vascular cell signaling, relatively little is known of the complexities of the in situ arrangement between specific ECM proteins and arteriolar SMCs. Given the above examples, it is likely that the structural arrangement of the vessel wall ECM proteins, particularly at the microvascular level, has an impact on how local mechanical forces are transmitted, sensed, and responded to and, ultimately, how effectively a vessel is able to alter its diameter. Conclusion-TheseAdding to difficulties in understanding the complexity of the extracellular components of the vessel wall is an apparent regional heterogeneity. Importantly, regional differences involve both variation in matrix composition and structural arrangement. Thus, in contrast to skeletal muscle arterioles...

Key points• The plasma membrane large-conductance Ca 2+ -activated, K + channel (BK Ca ) is a major ion channel contributing to the regulation of membrane potential.• Activation of large-conductance Ca 2+ -activated K + channel by both depolarization and increased intracellular Ca 2+ results in hyperpolarization that acts to limit agonist and mechanically induced vasoconstriction in small arteries.• Using patch-clamp techniques we demonstrate that regional differences exist in how BK Ca is regulated, particularly with respect to its Ca 2+ sensitivity.• Using single-channel recordings and siRNA to manipulate protein subunit expression, it is argued that the β1-subunit plays a more dominant role in cerebral blood vessels as compared with small arteries from skeletal muscle.• Subtle differences in the regulation of membrane potential in different vascular beds allow local blood flow and pressure to be closely adapted to the tissue's metabolic needs.Abstract β1-Subunits enhance the gating properties of large-conductance Ca 2+ -activated K + channels (BK Ca ) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), β1-subunits are vital in coupling SR-generated Ca 2+ sparks to BK Ca activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BK Ca activity due to apparent differences in the functional β1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. β1 modulation of the α-subunit Ca 2+ sensitivity was studied using patch-clamp techniques. BK Ca channel normalized open probability (NP o ) versus membrane potential (V m ) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca 2+ was raised from 0.5 to 100 μM. Calculated V 1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 μM Ca 2+ i to −89 ± 9 mV at 100 μM Ca VSMCs. Functionally, this leads both to higher Ca 2+ sensitivity and NP o for BK Ca channels in the cerebral vasculature relative to that of skeletal muscle.

Elastin distribution within the walls of resistance vessels is complex, with discrete fibers varying in orientation across the adventitia and media as well as the more sheet‐like structure of the internal elastic lamina (IEL). These structural patterns likely subserve specific functional properties including mechanosensing, control of forces, cellular positioning, and communication between vascular cells. To understand the impact of elastin on small artery function we initiated analyses of mRNA level expression patterns measured by qPCR, and protein distribution determined by fluorescence 3D confocal imaging. Studies were performed on 2nd order mesenteric arteries from rats aged 3, 7, 11, 14 and 19 days as well as 2 mths. Elastin mRNA peaked at day 11 before declining at later times. During the postnatal period, the elastin component of the IEL of mesenteric vessels accumulated/matured progressing from a punctate/fibrous appearance into a continuous sheet. Analysis of adventitial and medial elastin during this period showed accumulation of fibers as well as re‐organization of fiber orientation. Collectively these data show marked remodeling and development of elastin networks in the walls of small arteries as they mature. The impact of elastin remodeling on vessel function, and its relationship to concurrent changes in hemodynamics, require further study. (*Co‐first authors; NIH HL095486 to GAM)

Recent studies showed an intricate arrangement of elastin fibers in the walls of small arteries as well as marked differences in elastin content and distribution of cerebral vessels compared to those of mesentery or cremaster muscle. The present studies extended these observations to examine age‐related changes in elastin mRNA expression. Total RNA was extracted from small cerebral and mesenteric arteries of Sprague‐Dawley rats aged 3, 7, 11, 19 days, 2 mths and 2 yrs. Elastin mRNA expression levels were determined using qPCR. Relative expression levels were quantified using the 2−ΔΔCt method. Elastin mRNA levels for both vessel types increased from day 3 to a peak at 11 days after which levels declined throughout life. Peak levels of elastin mRNA expression were significantly greater in mesenteric compared to cerebral vessels (relative expression 24.23±2.90 and 14.78±1.23) and remained elevated at later time‐points. Differences in mRNA expression between cerebral and mesenteric vessels were consistent with earlier observations showing more elastin protein in the adventitia and media of mesenteric arteries. In addition to the temporal pattern of mRNA expression, the data show heterogeneity in magnitude of elastin expression in resistance artery walls from differing vascular beds. Such differences may impact function through their effects on the mechanical environment of the vessel wall. (Supp NIH HL095486)