Biophysical Induction of Vascular Smooth Muscle Cell Podosomes

Vascular smooth muscle cell (VSMC) migration and matrix degradation occurs with intimal hyperplasia associated with atherosclerosis, vascular injury, and restenosis. One proposed mechanism by which VSMCs degrade matrix is through the use of podosomes, transient actin-based structures that are thought to play a role in extracellular matrix degradation by creating localized sites of matrix metalloproteinase (MMP) secretion. To date, podosomes in VSMCs have largely been studied by stimulating cells with phorbol esters, such as phorbol 12,13-dibutyrate (PDBu), however little is known about the physiological cues that drive podosome formation. We present the first evidence that physiological, physical stimuli mimicking cues present within the microenvironment of diseased arteries can induce podosome formation in VSMCs. Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells. Moreover, wounding using a scratch assay induces podosomes at the leading edge of VSMCs. Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor. Together, these data indicate that physical cues can induce podosome formation in VSMCs.     

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.     

Effects of NS1608 on MaxiK Channels in Smooth Muscle Cells from Urinary Bladder

Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca²⁺-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 μm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels ∼100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 μm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence. Received: 19 July/Revised: 20 September 1999     

Role of Mitogen-Activated Protein Kinases in Stimulation of Smooth Muscle Cell Migration by Urokinase

The urokinase-type plasminogen activator, or urokinase, stimulates proliferation, adhesion, and migration of different type cells both due to its proteolytic activity and by activation of intracellular transduction pathways after interaction with the external cell surface. It is suggested that activation of p42/p44^erk1,2 MAP-kinases in response to specific receptor binding to the urokinase N-terminal domain is the key event in initiation of cell migration. However, we have found that the central kringle-domain of urokinase has its own target on the cell surface, and that its binding causes a migration response of human smooth muscle cells (SMCs). In the present study, we have shown that the urokinase kringle-domain is required to activate the p38 MAP kinase cascade, and that its inhibition leads to suppression of the migration response of SMCs. On the contrary, stimulation of the p42/p44^erk1,2 MAP-kinase cascade is determined only by proteolytic activity of urokinase and does not depend on its binding to SMCs. Selective inhibition of the p42/p44^erk1,2 MAP-kinase cascade produced a depression of the SMC migration induced by catalytically active urokinase, but did not affect the migration induced by non-active urokinase. It is concluded that binding of the urokinase kringle-domain to a yet unidentified target at the SMC surface is required for activation of the p38 MAP-kinase cascade and of the cell migration. Urokinase was shown to stimulate phosphorylation and activation of regulatory light myosin chains that are required to increase the cytoskeleton dynamics and SMC motility. The participation of p42/p44^erk1,2 and p38 MAP-kinase cascade in the realization of this effect is discussed.     

L-Type Ca2+ Channel Sparklets Revealed by TIRF Microscopy in Mouse Urinary Bladder Smooth Muscle

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca²⁺, referred to as Ca²⁺ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca²⁺ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca²⁺ (0.035±0.01 vs. 0.23±0.07 Hz/mm²; P<0.05). Co-loading of smooth muscle strips with the slow Ca²⁺ chelator EGTA-AM (10 mM) confirmed that Ca²⁺ sparklets are restricted to the cell membrane. Ca²⁺ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm²; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm²; P<0.05). Ca²⁺ sparklets were unaffected by inhibition of P2X₁ receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca²⁺ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm²; P<0.05), demonstrating that Ca²⁺ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca²⁺ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman''s rank correlation 0.2, P>0.05), implying that Ca²⁺ store mediated signals regulate Ca²⁺ sparklets. Under control conditions, inhibition of store operated Ca²⁺ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca²⁺ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca²⁺ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca²⁺ concentrations.     

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background

Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood.

Principal Findings

In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize.

Conclusions

Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.     

Resveratrol Induces Vascular Smooth Muscle Cell Differentiation through Stimulation of SirT1 and AMPK

Phenotypic plasticity in vascular smooth muscle cells (VSMC) is necessary for vessel maintenance, repair and adaptation to vascular changes associated with aging. De-differentiated VSMC contribute to pathologies including atherosclerosis and intimal hyperplasia. As resveratrol has been reported to have cardio- protective effects, we investigated its role in VSMC phenotypic modulation. We demonstrated the novel finding that resveratrol promoted VSMC differentiation as measured by contractile protein expression, contractile morphology and contraction in collagen gels. Resveratrol induced VSMC differentiation through stimulation of SirT1 and AMPK. We made the novel finding that low or high dose resveratrol had an initially different mechanism on induction of differentiation. We found that low dose resveratrol stimulated differentiation through SirT1-mediated activation of AKT, whereas high dose resveratrol stimulated differentiation through AMPK-mediated inhibition of the mTORC1 pathway, allowing activation of AKT. The health effects of resveratrol in cardiovascular diseases, cancer and longevity are an area of active research. We have demonstrated a supplemental avenue where-by resveratrol may promote health by maintaining and enhancing plasticity of the vasculature.     

Defining the Role of Vascular Smooth Muscle Cell Apoptosis in Atherosclerosis

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodelling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. Using transgenic mice with selective induction of VSMC apoptosis, a recent study has precisely determined the direct consequences of VSMC apoptosis in both normal vessels and atherosclerotic plaques. Surprisingly, normal arteries can withstand huge cell losses with little change in active or passive properties. Normal vessels demonstrate highly efficient clearance of apoptotic bodies, even in the absence of professional phagocytes. In contrast, VSMC apoptosis alone is sufficient to induce multiple features of vulnerability to rupture in plaques, identifying VSMC apoptosis as a critical process determining plaque stability.     

A Biophysical Analysis of Mitochondrial Movement: Differences Between Transport in Neuronal Cell Bodies Versus Processes

There is an increasing interest in factors that can impede cargo transport by molecular motors inside the cell. Although potentially relevant (Yi JY, Ori‐McKenney KM, McKenney RJ, Vershinin M, Gross SP, Vallee RB. High‐resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high‐load axonal transport. J Cell Biol 2011;195:193–201), the importance of cargo size and subcellular location has received relatively little attention. Here we address these questions taking advantage of the fact that mitochondria – a common cargo – in Drosophila neurons exhibit a wide distribution of sizes. In addition, the mitochondria can be genetically marked with green fluorescent protein (GFP) making it possible to visualize and compare their movement in the cell bodies and in the processes of living cells. Using total internal reflection microscopy coupled with particle tracking and analysis, we quantified the transport properties of GFP‐positive mitochondria as a function of their size and location. In neuronal cell bodies, we find little evidence for significant opposition to motion, consistent with a previous study on lipid droplets (Shubeita GT, Tran SL, Xu J, Vershinin M, Cermelli S, Cotton SL, Welte MA, Gross SP. Consequences of motor copy number on the intracellular transport of kinesin‐1‐driven lipid droplets. Cell 2008;135:1098–1107). However, in the processes, we observe an inverse relationship between the mitochondrial size and velocity and the run distances. This can be ameliorated via hypotonic treatment to increase process size, suggesting that motor‐mediated movement is impeded in this more‐confined environment. Interestingly, we also observe local mitochondrial accumulations in processes but not in cell bodies. Such accumulations do not completely block the transport but do increase the probability of mitochondria–mitochondria interactions. They are thus particularly interesting in relation to mitochondrial exchange of elements.      

自发性高血压大鼠血管平滑肌及心肌内成纤维细胞生长特性的研究

目的　探讨自发性高血压大鼠(spontaneously hypertensive rats, SHR)主动脉平滑肌细胞(aorta smooth muscle cell, ASMC)与心肌内成纤维细胞(cardiac fibroblast, CFB)在体外培养生长的特性。方法　16周龄自发性高血压大鼠(SHR)和正常大鼠(WKY)测血压后处死,取心脏与胸主动脉,组织块法分别培养CFB与ASMC)。分别观察(1)CFB     

Cytoskeletal and Cytocontractile Protein Composition of Smooth Muscle Cells in Developing and Obstructed Rabbit Bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, α-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM α-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

An Anti-Ubiquitin Antibody Response in Transitional Cell Carcinoma of the Urinary Bladder

Background

To use combinatorial epitope mapping (“fingerprinting”) of the antibody response to identify targets of the humoral immune response in patients with transitional cell carcinoma (TCC) of the bladder.

Methods

A combinatorial random peptide library was screened on the circulating pool of immunoglobulins purified from an index patient with a high risk TCC (pTa high grade plus carcinoma in situ) to identify corresponding target antigens. A patient cohort was investigated for antibody titers against ubiquitin.

Results

We selected, isolated, and validated an immunogenic peptide motif from ubiquitin as a dominant epitope of the humoral response. Patients with TCC had significantly higher antibody titers against ubiquitin than healthy donors (p<0.007), prostate cancer patients (p<0.0007), and all patients without TCC taken together (p<0.0001). Titers from superficial tumors were not significantly different from muscle invasive tumors (p = 0.0929). For antibody response against ubiquitin, sensitivity for detection of TCC was 0.44, specificity 0.96, positive predictive value 0.96 and negative predictive value 0.41. No significant titer changes were observed during the standard BCG induction immunotherapy.

Conclusions

This is the first report to demonstrate an anti-ubiquitin antibody response in patients with TCC. Although sensitivity of antibody production was low, a high specificity and positive predictive value make ubiquitin an interesting candidate for further diagnostic and possibly immune modulating studies.     

Induction of Smooth Muscle Cell Phenotype in Cultured Human Prostatic Stromal Cells

Stromal cells are key regulators of growth and differentiation in the adult human prostate. Alterations in the stroma are believed to initiate the development of benign prostatic hyperplasia, and stromal–epithelial interactions may have a role in malignant progression. The prostatic stroma is composed of two major cell types, smooth muscle cells and fibroblasts. Cell cultures from the prostatic stroma have been established by several investigators, but the phenotype of these cells has not been extensively characterized and it is not clear whether they are fibroblastic or smooth muscle-like. In this study, the response of stromal cells cultured from normal prostatic tissues to transforming growth factor-β (TGFβ) was investigated. We confirmed a previous report that TGFβ inhibited the growth of prostatic stromal cells in serum-containing medium, and showed that inhibition also occurred in serum-free medium. Growth inhibition by TGFβ was irreversible after 24 to 72 h of exposure. In the absence of TGFβ, cells were fibroblastic and expressed vimentin and fibronectin but little α-smooth muscle actin. After 3 days of exposure to 1 ng/ml of TGFβ, the majority of cells expressed α-smooth muscle actin and desmin, as demonstrated by immunocytochemistry and immunoblot analysis. This effect was specific and α-smooth muscle actin was not induced by two other growth-inhibitory factors, retinoic acid or 1,25-dihydroxyvitamin D₃. These results suggest that TGFβ is an important regulator of growth and differentiation of prostatic stromal cells and that a smooth muscle cell phenotype is promoted in the presence of TGFβ.