Reduced stiffness and augmented traction force in type 2 diabetic coronary microvascular smooth muscle.

Type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness and reduced coronary blood flow in both mice and pig models. Since reduced stiffness does not appear to be due to functional changes in the extracellular matrix, this study tested the hypothesis that decreased CRM stiffness in T2DM is due to reduced vascular smooth muscle cell (VSMC) stiffness, which impacts the traction force generated by VSMCs. Atomic force microscopy (AFM) and traction force microscopy (TFM) were conducted on primary low-passage CRM VSMCs from normal Db/db and T2DM db/db mice in addition to low-passage normal and T2DM deidentified human coronary VSMCs. Elastic modulus was reduced in T2DM mouse and human coronary VSMCs compared with normal (mouse: Db/db 6.84 ± 0.34 kPa vs. db/db 4.70 ± 0.19 kPa, P < 0.0001; human: normal 3.59 ± 0.38 kPa vs. T2DM 2.61 ± 0.35 kPa, P = 0.05). Both mouse and human T2DM coronary microvascular VSMCs were less adhesive to fibronectin compared with normal. T2DM db/db coronary VSMCs generated enhanced traction force by TFM (control 692 ± 67 Pa vs. db/db 1,507 ± 207 Pa; P < 0.01). Immunoblot analysis showed that T2DM human coronary VSMCs expressed reduced ß1-integrin and elevated ß3-integrin (control 1.00 ± 0.06 vs. T2DM 0.62 ± 0.14, P < 0.05 and control 1.00 ± 0.49 vs. T2DM 3.39 ± 1.05, P = 0.06, respectively). These data show that T2DM coronary VSMCs are less stiff and less adhesive to fibronectin but are able to generate enhanced force, corroborating previously published computational findings that decreasing cellular stiffness increases the cells' ability to generate higher traction force.NEW & NOTEWORTHY We show here that a potential causative factor for reduced diabetic coronary microvascular stiffness is the direct reduction in coronary vascular smooth muscle cell stiffness. These cells were also able to generate enhanced traction force, validating previously published computational models. Collectively, these data show that smooth muscle cell stiffness can be a contributor to overall tissue stiffness in the coronary microcirculation, and this may be a novel area of interest for therapeutic targets.

Switching behaviour in vascular smooth muscle cell-matrix adhesion during oscillatory loading.

Integrins regulate mechanotransduction between smooth muscle cells (SMCs) and the extracellular matrix (ECM). SMCs resident in the walls of airways or blood vessels are continuously exposed to dynamic mechanical forces due to breathing or pulsatile blood flow. However, the resulting effects of these forces on integrin dynamics and associated cell-matrix adhesion are not well understood. Here we present experimental results from atomic force microscopy (AFM) experiments, designed to study the integrin response to external oscillatory loading of varying amplitudes applied to live aortic SMCs, together with theoretical results from a mathematical model. In the AFM experiments, a fibronectin-coated probe was used cyclically to indent and retract from the surface of the cell. We observed a transition between states of firm adhesion and of complete detachment as the amplitude of oscillatory loading increased, revealed by qualitative changes in the force timecourses. Interestingly, for some of the SMCs in the experiments, switching behaviour between the two adhesion states is observed during single timecourses at intermediate amplitudes. We obtain two qualitatively similar adhesion states in the mathematical model, where we simulate the cell, integrins and ECM as an evolving system of springs, incorporating local integrin binding dynamics. In the mathematical model, we observe a region of bistability where both the firm adhesion and detachment states can occur depending on the initial adhesion state. The differences are seen to be a result of mechanical cooperativity of integrins and cell deformation. Switching behaviour is a phenomenon associated with bistability in a stochastic system, and bistability in our deterministic mathematical model provides a potential physical explanation for the experimental results. Physiologically, bistability provides a means for transient mechanical stimuli to induce long-term changes in adhesion dynamics-and thereby the cells' ability to transmit force-and we propose further experiments for testing this hypothesis.

Endothelial Mineralocorticoid Receptor Mediates Diet-Induced Aortic Stiffness in Females.

RATIONALE: Enhanced activation of the mineralocorticoid receptors (MRs) in cardiovascular tissues increases oxidative stress, maladaptive immune responses, and inflammation with associated functional vascular abnormalities. We previously demonstrated that consumption of a Western diet (WD) for 16 weeks results in aortic stiffening, and that these abnormalities were prevented by systemic MR blockade in female mice. However, the cell-specific role of endothelial cell MR (ECMR) in these maladaptive vascular effects has not been explored. OBJECTIVE: We hypothesized that specific deletion of the ECMR would prevent WD-induced increases in endothelial sodium channel activation, reductions in bioavailable nitric oxide, increased vascular remodeling, and associated increases in vascular stiffness in females. METHODS AND RESULTS: Four-week-old female ECMR knockout and wild-type mice were fed either mouse chow or WD for 16 weeks. WD feeding resulted in aortic stiffness and endothelial dysfunction as determined in vivo by pulse wave velocity and ex vivo by atomic force microscopy, and wire and pressure myography. The WD-induced aortic stiffness was associated with enhanced endothelial sodium channel activation, attenuated endothelial nitric oxide synthase activation, increased oxidative stress, a proinflammatory immune response and fibrosis. Conversely, cell-specific ECMR deficiency prevented WD-induced aortic fibrosis and stiffness in conjunction with reductions in endothelial sodium channel activation, oxidative stress and macrophage proinflammatory polarization, restoration of endothelial nitric oxide synthase activation. CONCLUSIONS: Increased ECMR signaling associated with consumption of a WD plays a key role in endothelial sodium channel activation, reduced nitric oxide production, oxidative stress, and inflammation that lead to aortic remodeling and stiffness in female mice.

N-Cadherin, a novel and rapidly remodelling site involved in vasoregulation of small cerebral arteries.

KEY POINTS: N-cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery. The formation of N-cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10-5  m) and ACh (10-5  m). N-cadherin-coated beads were able to induce clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10-5  m) or sodium nitroprusside (10-5  m) induced a significant increase or decrease in the N-cadherin-EGFP clustering, respectively. Application of pulling force (∼1 nN) to the N-cadherin-coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling. ABSTRACT: N-cadherin is the major cell-cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N-cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N-cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90 mmHg, both the density and the average size of N-cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10-5  m) induced a significant increase of N-cadherin AJ density at 50 mmHg, whereas vasodilatation induced by ACh (10-5  m) was accompanied by a significant decrease in density and size of N-cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano-responsive properties of N-cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N-cadherin-coated microbead (5 µm) induced a progressive clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1 nN) to the N-cadherin-coated-beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10-5  m) or sodium nitroprusside (10-5  m) induced a significant increase or decrease of the N-cadherin-EGFP clustering, respectively. These observations provide compelling evidence that N-cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N-cadherin AJs in vasomotor regulation.

Nonenzymatic glycation interferes with fibronectin-integrin interactions in vascular smooth muscle cells.

OBJECTIVE: We aimed to investigate whether advanced nonenzymatic glycation of the ECM protein, fibronectin, impacts its normal integrin-mediated interaction with arteriolar VSMC. METHODS: AFM was performed on cultured VSMC from rat cremaster arterioles to study native and glycated fibronectin (FN and gFN) interactions with cellular integrins. AFM probes were functionalized with FN or gFN or with native or glycated albumin (gAlb) as controls. RESULTS: VSMC showed increased adhesion probability to gFN (72.9±3.5%) compared with native FN (63.0±1.6%). VSMC similarly showed increased probability of adhesion (63.8±1.7%) to gAlb compared with native Alb (40.1±4.7%). Adhesion of native FN to VSMC was α5 and ß1 integrin dependent whereas adhesion of gFN to VSMC was integrin independent. The RAGE-selective inhibitor, FPS-ZM1, blocked gFN (and gAlb) adhesion, suggesting that adhesion of glycated proteins was RAGE dependent. Interaction of FN with VSMC was not altered by soluble gFN while soluble native FN did not inhibit adhesion of gFN to VSMC. In contrast, gAlb inhibited adhesion of gFN to VSMC in a concentration-dependent manner. CONCLUSIONS: Glycation of FN shifts the nature of cellular adhesion from integrin- to RAGE-dependent mechanisms.

Extracellular Matrix Disarray as a Mechanism for Greater Abdominal Versus Thoracic Aortic Stiffness With Aging in Primates.

OBJECTIVE: Increased vascular stiffness is central to the pathophysiology of aging, hypertension, diabetes mellitus, and atherosclerosis. However, relatively few studies have examined vascular stiffness in both the thoracic and the abdominal aorta with aging, despite major differences in anatomy, embryological origin, and relation to aortic aneurysm. APPROACH AND RESULTS: The 2 other unique features of this study were (1) to study young (9±1 years) and old (26±1 years) male monkeys and (2) to study direct and continuous measurements of aortic pressure and thoracic and abdominal aortic diameters in conscious monkeys. As expected, aortic stiffness, ß, was increased P<0.05, 2- to 3-fold, in old versus young thoracic aorta and augmented further with superimposition of acute hypertension with phenylephrine. Surprisingly, stiffness was not greater in old thoracic aorta than in young abdominal aorta. These results can be explained, in part, by the collagen/elastin ratio, but more importantly, by disarray of collagen and elastin, which correlated best with vascular stiffness. However, vascular smooth muscle cell stiffness was not different in thoracic versus abdominal aorta in either young or old monkeys. CONCLUSIONS: Thus, aortic stiffness increases with aging as expected, but the most severe increases in aortic stiffness observed in the abdominal aorta is novel, where values in young monkeys equaled, or even exceeded, values of thoracic aortic stiffness in old monkeys. These results can be explained by alterations in collagen/elastin ratio, but even more importantly by collagen and elastin disarray.

Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles.

KEY POINTS: Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1 R), causes a concentration-dependent inhibition of pressure-dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein-coupled receptor. Mechanoactivation of the AT1 R occurs independently of local angiotensin II production and the type 2 angiotensin receptor. Mechanoactivation of the AT1 R stimulates actin polymerization by a protein kinase C-dependent mechanism, but independently of a change in intracellular Ca2+ . Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1 R. ABSTRACT: The Gq/11 protein-coupled angiotensin II type 1 receptor (AT1 R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1 R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1 R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first-order cremaster muscle arterioles the AT1 R inhibitor candesartan (10-7 -10-5  m) showed partial but concentration-dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure-diameter relationship over the intraluminal pressure range, 30-110 mmHg. Pressure-induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura-2) were similar in the absence or presence of candesartan, indicating that AT1 R-mediated myogenic constriction relies on Ca2+ -independent downstream signalling. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G-actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1 R in arterioles and suggest that mechanically activated AT1 R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure-induced vasoconstriction.

Brief serotonin exposure initiates arteriolar inward remodeling processes in vivo that involve transglutaminase activation and actin cytoskeleton reorganization.

Inward remodeling of the resistance vasculature is strongly associated with life-threatening cardiovascular events. Previous studies have demonstrated that both actin polymerization and the activation of transglutaminases mediate early stages of the transition from a structurally normal vessel to an inwardly remodeled one. Ex vivo studies further suggest that a few hours of exposure to vasoconstrictor agonists induces inward remodeling in the absence of changes in intraluminal pressure. Here we report that a short, 10-min, topical exposure to serotonin (5-HT) + N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME) was sufficient to initiate inward remodeling processes in rat cremasteric feed arterioles (100-200 µm lumen diameter), in vivo. Addition of the transglutaminase inhibitor, cystamine, blocked the in vivo remodeling. We further demonstrate that, in isolated arterioles, 5-HT + l-NAME activates transglutaminases and modulates the phosphorylation state of cofilin, a regulator of actin depolymerization. The 5-HT + l-NAME-induced remodeling process in isolated arterioles was also inhibited by an inhibitor of Lim Kinase, the kinase that phosphorylates and inactivates cofilin. Therefore, our results indicate that a brief vasoconstriction induced by 5-HT + l-NAME is able to reduce the passive structural diameter of arterioles through processes that are dependent on the activation of transglutaminases and Lim kinase, and the subsequent phosphorylation of cofilin.

Small artery mechanobiology: Roles of cellular and non-cellular elements.

Small arteries and their component cellular and non-cellular elements are continually subjected to, and interact with, mechanical forces. Such interactions are key in both short- and long-term adaptation of the structure and function of the microcirculation to its local environment and metabolic requirements. Following this brief introduction is a series of papers presented as a symposium (Small Artery Mechanobiology: Roles of Cellular and Non-Cellular Elements) at the World Congress for Microcirculation, Kyoto 2015.

Small Artery Elastin Distribution and Architecture-Focus on Three Dimensional Organization.

The distribution of ECM proteins within the walls of resistance vessels is complex both in variety of proteins and structural arrangement. In particular, elastin exists as discrete fibers varying in orientation across the adventitia and media as well as often resembling a sheet-like structure in the case of the IEL. Adding to the complexity is the tissue heterogeneity that exists in these structural arrangements. For example, small intracranial cerebral arteries lack adventitial elastin while similar sized arteries from skeletal muscle and intestinal mesentery exhibit a complex adventitial network of elastin fibers. With regard to the IEL, several vascular beds exhibit an elastin sheet with punctate holes/fenestrae while in others the IEL is discontinuous and fibrous in appearance. Importantly, these structural patterns likely sub-serve specific functional properties, including mechanosensing, control of external forces, mechanical properties of the vascular wall, cellular positioning, and communication between cells. Of further significance, these processes are altered in vascular disorders such as hypertension and diabetes mellitus where there is modification of ECM. This brief report focuses on the three-dimensional wall structure of small arteries and considers possible implications with regard to mechanosensing under physiological and pathophysiological conditions.

Dipeptidyl peptidase-4 inhibition with linagliptin prevents western diet-induced vascular abnormalities in female mice.

BACKGROUND: Vascular stiffening, a risk factor for cardiovascular disease, is accelerated, particularly in women with obesity and type 2 diabetes. Preclinical evidence suggests that dipeptidylpeptidase-4 (DPP-4) inhibitors may have cardiovascular benefits independent of glycemic lowering effects. Recent studies show that consumption of a western diet (WD) high in fat and simple sugars induces aortic stiffening in female C57BL/6J mice in advance of increasing blood pressure. The aims of this study were to determine whether administration of the DPP-4 inhibitor, linagliptin (LGT), prevents the development of aortic and endothelial stiffness induced by a WD in female mice. METHODS: C56Bl6/J female mice were fed a WD for 4 months. Aortic stiffness and ex vivo endothelial stiffness were evaluated by Doppler pulse wave velocity (PWV) and atomic force microscopy (AFM), respectively. In addition, we examined aortic vasomotor responses and remodeling markers via immunohistochemistry. Results were analyzed via 2-way ANOVA, p < 0.05 was considered as statistically significant. RESULTS: Compared to mice fed a control diet (CD), WD-fed mice exhibited a 24 % increase in aortic PWV, a five-fold increase in aortic endothelial stiffness, and impaired endothelium-dependent vasodilation. In aorta, these findings were accompanied by medial wall thickening, adventitial fibrosis, increased fibroblast growth factor 23 (FGF-23), decreased Klotho, enhanced oxidative stress, and endothelial cell ultrastructural changes, all of which were prevented with administration of LGT. CONCLUSIONS: The present findings support the notion that DPP-4 plays a role in development of WD-induced aortic stiffening, vascular oxidative stress, endothelial dysfunction, and vascular remodeling. Whether, DPP-4 inhibition could be a therapeutic tool used to prevent the development of aortic stiffening and the associated cardiovascular complications in obese and diabetic females remains to be elucidated.

Attenuated sarcomere lengthening of the aged murine left ventricle observed using two-photon fluorescence microscopy.

The Frank-Starling mechanism, whereby increased diastolic filling leads to increased cardiac output, depends on increasing the sarcomere length (Ls) of cardiomyocytes. Ventricular stiffness increases with advancing age, yet it remains unclear how such changes in compliance impact sarcomere dynamics in the intact heart. We developed an isolated murine heart preparation to monitor Ls as a function of left ventricular pressure and tested the hypothesis that sarcomere lengthening in response to ventricular filling is impaired with advanced age. Mouse hearts isolated from young (3-6 mo) and aged (24-28 mo) C57BL/6 mice were perfused via the aorta under Ca(2+)-free conditions with the left ventricle cannulated to control filling pressure. Two-photon imaging of 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium fluorescence was used to monitor t-tubule striations and obtain passive Ls between pressures of 0 and 40 mmHg. Ls values (in µm, aged vs. young, respectively) were 2.02 ± 0.04 versus 2.01 ± 0.02 at 0 mmHg, 2.13 ± 0.04 versus 2.23 ± 0.02 at 5 mmHg, 2.21 ± 0.03 versus 2.27 ± 0.03 at 10 mmHg, and 2.28 ± 0.02 versus 2.36 ± 0.01 at 40 mmHg, indicative of impaired sarcomere lengthening in aged hearts. Atomic force microscopy nanoindentation revealed that intact cardiomyocytes enzymatically isolated from aged hearts had increased stiffness compared with those of young hearts (elastic modulus: aged, 41.9 ± 5.8 kPa vs. young, 18.6 ± 3.3 kPa; P = 0.006). Impaired sarcomere lengthening during left ventricular filling may contribute to cardiac dysfunction with advancing age by attenuating the Frank-Starling mechanism and reducing stroke volume.

Vasoactive agonists exert dynamic and coordinated effects on vascular smooth muscle cell elasticity, cytoskeletal remodelling and adhesion.

In this study, we examined the ability of vasoactive agonists to induce dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion, and tested the hypothesis that these events are coordinated with rapid remodelling of the cortical cytoskeleton. Real-time measurement of cell elasticity was performed with atomic force microscopy (AFM) and adhesion was assessed with AFM probes coated with fibronectin (FN). Temporal data were analysed using an Eigen-decomposition method. Elasticity in VSMCs displayed temporal oscillations with three components at approximately 0.001, 0.004 and 0.07 Hz, respectively. Similarly, adhesion displayed a similar oscillatory pattern. Angiotensin II (ANG II, 10(-6) M) increased (+100%) the amplitude of the oscillations, whereas the vasodilator adenosine (ADO, 10(-4) M) reduced oscillation amplitude (-30%). To test whether the oscillatory changes were related to the architectural alterations in cortical cytoskeleton, the topography of the submembranous actin cytoskeleton (100-300 nm depth) was acquired with AFM. These data were analysed to compare cortical actin fibre distribution and orientation before and after treatment with vasoactive agonists. The results showed that ANG II increased the density of stress fibres by 23%, while ADO decreased the density of the stress fibres by 45%. AFM data were supported by Western blot and confocal microscopy. Collectively, these observations indicate that VSMC cytoskeletal structure and adhesion to the extracellular matrix are dynamically altered in response to agonist stimulation. Thus, vasoactive agonists probably invoke unique mechanisms that dynamically alter the behaviour and structure of both the VSMC cytoskeleton and focal adhesions to efficiently support the normal contractile behaviour of VSMCs.

N-cadherin, a vascular smooth muscle cell-cell adhesion molecule: function and signaling for vasomotor control.

Cell-cell adhesion complexes are increasingly recognized as an important cell-signaling site, similar to integrin-extracellular matrix FA. Furthermore, cell-cell adhesions are involved in the regulation of multi-cellular/tissue organization and organ, tissue, and cellular level functional behavior. Although N-cadherin is the major cell-cell adhesion molecule in VSM, only limited studies have been undertaken to understand its function in VSM. In contrast, N-cadherin signaling and functions have been extensively studied in neurons, fibroblasts, and myocytes, as well as in the context of epithelial-mesenchymal-transitions. Increasing evidence has indicated that N-cadherin-mediated cell-cell adhesions are important for tissue integrity and cell proliferation. Relevant to VSM, N-cadherin's role in actin cytoskeleton organization and contraction, as well as its role in regulation of Rho family GTPases are of particular interest. This article briefly reviews the fundamentals of N-cadherin biology that help shape our current understanding of its function and signaling mechanisms. In particular, attention is given to applications of this knowledge to VSM. The review points to the need for more research effort that is directed at understanding the role of N-cadherins in the regulation of vascular function.

The role of reactive oxygen species in microvascular remodeling.

The microcirculation is a portion of the vascular circulatory system that consists of resistance arteries, arterioles, capillaries and venules. It is the place where gases and nutrients are exchanged between blood and tissues. In addition the microcirculation is the major contributor to blood flow resistance and consequently to regulation of blood pressure. Therefore, structural remodeling of this section of the vascular tree has profound implications on cardiovascular pathophysiology. This review is focused on the role that reactive oxygen species (ROS) play on changing the structural characteristics of vessels within the microcirculation. Particular attention is given to the resistance arteries and the functional pathways that are affected by ROS in these vessels and subsequently induce vascular remodeling. The primary sources of ROS in the microcirculation are identified and the effects of ROS on other microcirculatory remodeling phenomena such as rarefaction and collateralization are briefly reviewed.

Novel role for vinculin in ventricular myocyte mechanics and dysfunction.

Vinculin (Vcl) plays a key structural role in ventricular myocytes that, when disrupted, can lead to contractile dysfunction and dilated cardiomyopathy. To investigate the role of Vcl in myocyte and myocardial function, cardiomyocyte-specific Vcl knockout mice (cVclKO) and littermate control wild-type mice were studied with transmission electron microscopy (TEM) and in vivo magnetic resonance imaging (MRI) tagging before the onset of global ventricular dysfunction. MRI revealed significantly decreased systolic strains transverse to the myofiber axis in vivo, but no changes along the muscle fibers or in fiber tension in papillary muscles from heterozygous global Vcl null mice. Myofilament lattice spacing from TEM was significantly greater in cVclKO versus wild-type hearts fixed in the unloaded state. AFM in Vcl heterozygous null mouse myocytes showed a significant decrease in membrane cortical stiffness. A multiscale computational model of ventricular mechanics incorporating cross-bridge geometry and lattice mechanics showed that increased transverse systolic stiffness due to increased lattice spacing may explain the systolic wall strains associated with Vcl deficiency, before the onset of ventricular dysfunction. Loss of cardiac myocyte Vcl may decrease systolic transverse strains in vivo by decreasing membrane cortical tension, which decreases transverse compression of the lattice thereby increasing interfilament spacing and stress transverse to the myofibers.

Increased vascular smooth muscle cell stiffness: a novel mechanism for aortic stiffness in hypertension.

Increased vascular stiffness is fundamental to hypertension, and its complications, including atherosclerosis, suggest that therapy should also be directed at vascular stiffness, rather than just the regulation of peripheral vascular resistance. It is currently held that the underlying mechanisms of vascular stiffness in hypertension only involve the extracellular matrix and endothelium. We hypothesized that increased large-artery stiffness in hypertension is partly due to intrinsic mechanical properties of vascular smooth muscle cells. After confirming increased arterial pressure and aortic stiffness in spontaneously hypertensive rats, we found increased elastic stiffness of aortic smooth muscle cells of spontaneously hypertensive rats compared with Wistar-Kyoto normotensive controls using both an engineered aortic tissue model and atomic force microscopy nanoindentation. Additionally, we observed different temporal oscillations in the stiffness of vascular smooth muscle cells derived from hypertensive and control rats, suggesting that a dynamic component to cellular elastic stiffness is altered in hypertension. Treatment with inhibitors of vascular smooth muscle cell cytoskeletal proteins reduced vascular smooth muscle cell stiffness from hypertensive and control rats, suggesting their participation in the mechanism. This is the first study demonstrating that stiffness of individual vascular smooth muscle cells mediates vascular stiffness in hypertension, a novel concept, which may elucidate new therapies for hypertension and for vascular stiffness.

Atomic force microscopy to characterize binding properties of α7-containing nicotinic acetylcholine receptors on neurokinin-1 receptor-expressing medullary respiratory neurons.

In the present study, we used atomic force microscopy (AFM) to examine the ligand-binding properties of α7-containing nicotinic acetylcholine receptors (nAChRs) expressed on neurons from the ventral respiratory group. We also determined the effect of acute and prolonged exposure to nicotine on the binding probability of nAChRs. Neurons from neonatal (postnatal day 5-10) and juvenile rats (3-4 weeks old) were cultured. Internalization of Alexa Fluor 488-conjugated substance P was used to identify respiratory neurons that expressed the neurokinin-1 receptor (NK1-R), a recognized marker of ventral respiratory group neurons. To assess functional changes in nAChRs, AFM probes conjugated with anti-α7 subunit nAChR antibody were used to interact cyclically with the surface of the soma of NK1-R-positive neurons. Measurements were made of the frequency of antibody adhesion to the α7 receptor subunit and of the detachment forces between the membrane-attached receptor and the AFM probe tip. Addition of α-bungarotoxin (a specific antagonist of α7 subunit-containing nAChRs) to the cell bath produced a 69% reduction in binding to the α7 subunit (P < 0.05, n = 10), supporting specificity of binding. Acute exposure to nicotine (1 µM added to culture media) produced an 80% reduction in nAChR antibody binding to the α7 subunit (P < 0.05, n = 9). Prolonged incubation (72 h) of the cell culture in nicotine significantly reduced α7 binding in a concentration-dependent manner. Collectively, these findings demonstrate that AFM is a sensitive tool for assessment of functional changes in nAChRs expressed on the surface of living NK1-R-expressing medullary neurons. Moreover, these data demonstrate that nicotine exposure decreases the binding probability of α7 subunit-containing nAChRs.

Postnatal development of extracellular matrix and vascular function in small arteries of the rat.

Introduction: Vascular extracellular matrix (ECM) is dominated by elastic fibers (elastin with fibrillin-rich microfibrils) and collagens. Current understanding of ECM protein development largely comes from studies of conduit vessels (e.g., aorta) while resistance vessel data are sparse. With an emphasis on elastin, we examined whether changes in postnatal expression of arteriolar wall ECM would correlate with development of local vasoregulatory mechanisms such as the myogenic response and endothelium-dependent dilation. Methods: Rat cerebral and mesenteric arteries were isolated at ages 3, 7, 11, 14, 19 days, 2 months, and 2 years. Using qPCR mRNA expression patterns were examined for elastin, collagen types I, II, III, IV, fibrillin-1, and -2, lysyl oxidase (LOX), and transglutaminase 2. Results: Elastin, LOX and fibrillar collagens I and III mRNA peaked at day 11-14 in both vasculatures before declining at later time-points. 3D confocal imaging for elastin showed continuous remodeling in the adventitia and the internal elastic lamina for both cerebral and mesenteric vessels. Myogenic responsiveness in cannulated cerebral arteries was detectable at day 3 with constriction shifted to higher intraluminal pressures by day 19. Myogenic responsiveness of mesenteric vessels appeared fully developed by day 3. Functional studies were performed to investigate developmental changes in endothelial-dependent dilation. Endothelial-dependent dilation to acetylcholine was less at day 3 compared to day 19 and at day 3 lacked an endothelial-derived hyperpolarizing factor component that was evident at day 19. Conclusion: Collectively, in the rat small artery structural remodeling and aspects of functional control continue to develop in the immediate postnatal period.

Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca(2+) signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca(2+) signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268-C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca(2+). Experiments were performed in low passage (p3∼10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 µm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800∼3000 pN) did not induce a Ca(2+) increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca(2+) increases. However, these Ca(2+) increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca(2+) using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca(2+) increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction leading to focal contraction that is independent of the need for a global increase in VSMC Ca(2+).