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.

Modification of Fibronectin by Non-Enzymatic Glycation Impairs K+ Channel Function in Rat Cerebral Artery Smooth Muscle Cells.

Fibronectin (FN) enhances K+ channel activity by integrin-mediated mechanisms. As vascular smooth muscle (VSM) K+ channels mediate vasodilation, we hypothesized that modification of fibronectin, via advanced non-enzymatic glycation, would alter signaling of this extracellular matrix protein through these channels. Bovine FN (1 mg/ml) was glycated (gFN) for 5 days using methylglyoxal (50 mM), and albumin was similarly glycated as a non-matrix protein control. VSM cells were isolated from rat cerebral arteries for measurement of macroscopic K+ channel activity using whole cell patch clamp methodology. Pharmacological inhibitors, iberiotoxin (0.1 µM) and 4-aminopyridine (0.1 mM), were used to identify contributions of large-conductance, Ca2+-activated, K+ channels and voltage-gated K+ channels, respectively. Compared with baseline, native FN enhanced whole cell K+ current in a concentration-dependent manner, whereas gFN inhibited basal current. Furthermore, native albumin did not enhance basal K+ current, but the glycated form (gAlb) caused inhibition. gFN was shown to impair both the Kv and BKCa components of total macroscopic K+ current. Anti-integrin α5 and ß1 antibodies attenuated the effects of both FN and gFN on macroscopic K+ current at +70 mV. Consistent with an action on BKCa activity, FN increased, whereas gFN decreased the frequency of spontaneous transient outward current (STOCs). In contrast, gAlb inhibited whole cell K+ current predominantly through Kv, showing little effect on STOCs. A function-blocking, anti-RAGE antibody partially reversed the inhibitory effects of gFN, suggesting involvement of this receptor. Further, gFN caused production of reactive oxygen species (ROS) by isolated VSMCs as revealed by the fluorescent indicator, DHE. Evoked ROS production was attenuated by the RAGE blocking antibody. Collectively, these studies identify ion channel-related mechanisms (integrin and ROS-mediated) by which protein glycation may modify VSMC function.

Changes in dynamics of tumor/endothelial cell adhesive interactions depending on endothelial cell growth state and elastic properties.

Cancer cell adhesion to the endothelium is a crucial process in hematogenous metastasis, but how the integrity of the endothelial barrier and endothelial cell (EC) mechanical properties influence the adhesion between metastatic cancer cells and the endothelium remain unclear. In the present study, we have measured the adhesion between single cancer cells and two types of ECs at various growth states and their mechanical properties (elasticity) using atomic force microscopy single cell force spectroscopy. We demonstrated that the EC stiffness increased and adhesion with cancer cells decreased, as ECs grew from a single cell to a confluent state and developed cell-cell contacts, but this was reversed when confluent cells returned to a single state in a scratch assay. Our results suggest that the integrity of the endothelial barrier is an important factor in reducing the ability of the metastatic tumor cells to adhere to the vascular endothelium, extravasate and lodge in the vasculature of a distant organ where secondary metastatic tumors would develop.

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.

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.

Temporal and molecular dynamics of human metastatic breast carcinoma cell adhesive interactions with human bone marrow endothelium analyzed by single-cell force spectroscopy.

Bone is a common site of metastasis for breast cancer and the mechanisms of metastasis are not fully elucidated. The purpose of our study was to characterize temporal and molecular dynamics of adhesive interactions between human breast cancer cells (HBCC) and human bone marrow endothelium (HBME) with piconewton resolution using atomic force microscopy (AFM). In adhesion experiments, a single breast cancer cell, MDA-MB-231 (MB231) or MDA-MB-435 (MB435) was attached to the AFM cantilever and brought into contact with a confluent HBME monolayer for different time periods (0.5 to 300 sec). The forces required to rupture individual molecular interactions and completely separate interacting cells were analyzed as measures of cell-cell adhesion. Adhesive interactions between HBME and either MB231 or MB435 cells increased progressively as cell-cell contact time was prolonged from 0.5 to 300 sec due to the time-dependent increase in the number and frequency of individual adhesive events, as well as to the involvement of stronger ligand-receptor interactions over time. Studies of the individual molecule involvement revealed that Thomsen-Friedenreich antigen (TF-Ag), galectin-3, integrin-ß1, and integrin-α3 are all contributing to HBCC/HBME adhesion to various degrees in a temporally defined fashion. In conclusion, cell-cell contact time enhances adhesion of HBCC to HBME and the adhesion is mediated, in part, by TF-Ag, galectin-3, integrin-α3, and integrin-ß1.

Applications of Atomic Force Microscopy for Adhesion Force Measurements in Mechanotransduction.

Adhesive interactions between living cells or ligand-receptor interactions can be studied at the molecular level using atomic force microscopy (AFM). Adhesion force measurements are performed with functionalized AFM probes. In order to measure single ligand-receptor interactions, a cantilever with a pyramidal tip is functionalized with a bio-recognized ligand (e.g., extracellular matrix protein). The ligand-functionalized probe is then brought into contact with a cell in culture to investigate adhesion between the respective probe-bound ligand and endogenously expressed cell surface receptors (e.g., integrins or other adhesion receptor). For experiments designed to examine cell-cell adhesions, a single cell is attached to a tipless cantilever which is then brought into contact with other cultured cells. Force curves are recorded to determine the forces necessary to rupture discrete adhesions between the probe-bound ligand and receptor, or to determine total adhesion force at cell-cell contacts. Here, we describe the procedures for measuring adhesions between (a) fibronectin and α5ß1 integrin, and (b) breast cancer cells and bone marrow endothelial cells.

Spontaneous oscillation in cell adhesion and stiffness measured using atomic force microscopy.

Atomic force microscopy (AFM) is an attractive technique for studying biomechanical and morphological changes in live cells. Using real-time AFM monitoring of cellular mechanical properties, spontaneous oscillations in cell stiffness and cell adhesion to the extracellular matrix (ECM) have been found. However, the lack of automated analytical approaches to systematically extract oscillatory signals, and noise filtering from a large set of AFM data, is a significant obstacle when quantifying and interpreting the dynamic characteristics of live cells. Here we demonstrate a method that extends the usage of AFM to quantitatively investigate live cell dynamics. Approaches such as singular spectrum analysis (SSA), and fast Fourier transform (FFT) were introduced to analyze a real-time recording of cell stiffness and the unbinding force between the ECM protein-decorated AFM probe and vascular smooth muscle cells (VSMCs). The time series cell adhesion and stiffness data were first filtered with SSA and the principal oscillatory components were isolated from the noise floor with the computed eigenvalue from the lagged-covariance matrix. Following the SSA, the oscillatory parameters were detected by FFT from the noise-reduced time series data sets and the sinusoidal oscillatory components were constructed with the parameters obtained by FFT.

A Calcium Mediated Mechanism Coordinating Vascular Smooth Muscle Cell Adhesion During KCl Activation.

Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca2+ ([Ca2+]i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-α5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an α5 integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-α5 antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca2+]i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca2+]i, which was measured using the Ca2+ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca2+]i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca2+]i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Angiotensin II Type 1 Receptor Mechanoactivation Involves RGS5 (Regulator of G Protein Signaling 5) in Skeletal Muscle Arteries: Impaired Trafficking of RGS5 in Hypertension.

Studies suggest that arteriolar pressure-induced vasoconstriction can be initiated by GPCRs (G protein-coupled receptors), including the AT1R (angiotensin II type 1 receptor). This raises the question, are such mechanisms regulated by negative feedback? The present studies examined whether RGS (regulators of G protein signaling) proteins in vascular smooth muscle cells are colocalized with the AT1R when activated by mechanical stress or angiotensin II and whether this modulates AT1R-mediated vasoconstriction. To determine whether activation of the AT1R recruits RGS5, an in situ proximity ligation assay was performed in primary cultures of cremaster muscle arteriolar vascular smooth muscle cells treated with angiotensin II or hypotonic solution in the absence or presence of candesartan (an AT1R blocker). Proximity ligation assay results revealed a concentration-dependent increase in trafficking/translocation of RGS5 toward the activated AT1R, which was attenuated by candesartan. In intact arterioles, knockdown of RGS5 enhanced constriction to angiotensin II and augmented myogenic responses to increased intraluminal pressure. Myogenic constriction was attenuated to a higher degree by candesartan in RGS5 siRNA-transfected arterioles, consistent with RGS5 contributing to downregulation of AT1R-mediated signaling. Further, translocation of RGS5 was impaired in vascular smooth muscle cells of spontaneously hypertensive rats. This is consistent with dysregulated (RGS5-mediated) AT1R signaling that could contribute to excessive vasoconstriction in hypertension. In intact vessels, candesartan reduced myogenic vasoconstriction to a greater extent in spontaneously hypertensive rats compared with controls. Collectively, these findings suggest that AT1R activation results in translocation of RGS5 toward the plasma membrane, limiting AT1R-mediated vasoconstriction through its role in Gq/11 protein-dependent signaling.

Uric acid promotes vascular stiffness, maladaptive inflammatory responses and proteinuria in western diet fed mice.

OBJECTIVE: Aortic vascular stiffness has been implicated in the development of cardiovascular disease (CVD) and chronic kidney disease (CKD) in obese individuals. However, the mechanism promoting these adverse effects are unclear. In this context, promotion of obesity through consumption of a western diet (WD) high in fat and fructose leads to excess circulating uric acid. There is accumulating data implicating elevated uric acid in the promotion of CVD and CKD. Accordingly, we hypothesized that xanthine oxidase(XO) inhibition with allopurinol would prevent a rise in vascular stiffness and proteinuria in a translationally relevant model of WD-induced obesity. MATERIALS/METHODS: Four-week-old C57BL6/J male mice were fed a WD with excess fat (46%) and fructose (17.5%) with or without allopurinol (125mg/L in drinking water) for 16weeks. Aortic endothelial and extracellular matrix/vascular smooth muscle stiffness was evaluated by atomic force microscopy. Aortic XO activity, 3-nitrotyrosine (3-NT) and aortic endothelial sodium channel (EnNaC) expression were evaluated along with aortic expression of inflammatory markers. In the kidney, expression of toll like receptor 4 (TLR4) and fibronectin were assessed along with evaluation of proteinuria. RESULTS: XO inhibition significantly attenuated WD-induced increases in plasma uric acid, vascular XO activity and oxidative stress, in concert with reductions in proteinuria. Further, XO inhibition prevented WD-induced increases in aortic EnNaC expression and associated endothelial and subendothelial stiffness. XO inhibition also reduced vascular pro-inflammatory and maladaptive immune responses induced by consumption of a WD. XO inhibition also decreased WD-induced increases in renal TLR4 and fibronectin that associated proteinuria. CONCLUSIONS: Consumption of a WD leads to elevations in plasma uric acid, increased vascular XO activity, oxidative stress, vascular stiffness, and proteinuria all of which are attenuated with allopurinol administration.

Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells.

Previous studies have shown that the aging kidney has a marked loss of α(E)-catenin in proximal tubular epithelium. α-Catenin, a key regulator of the actin cytoskeleton, interacts with a variety of actin-binding proteins. Cisplatin-induced loss of fascin2, an actin bundling protein, was observed in cells with a stable knockdown of α(E)-catenin (C2 cells), as well as in aging (24 mon), but not young (4 mon), kidney. Fascin2 co-localized with α-catenin and the actin cytoskeleton in NRK-52E cells. Knockdown of fascin2 increased the susceptibility of tubular epithelial cells to cisplatin-induced injury. Overexpression of fascin2 in C2 cells restored actin stress fibers and attenuated the increased sensitivity of C2 cells to cisplatin-induced apoptosis. Interestingly, fascin2 overexpression attenuated cisplatin-induced mitochondrial dysfunction and oxidative stress in C2 cells. These data demonstrate that fascin2, a putative target of α(E)-catenin, may play important role in preventing cisplatin-induced acute kidney injury.

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.

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.

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.

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.