Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells

During pregnancy, stretch of the uterus, imposed by the growing fetus, is an important signal for the induction of genes involved in the onset of labor. In this study, the expression of activator protein-1 (AP-1) family mRNAs in response to in vitro stretch was investigated in myometrial cells. Rat primary myometrial smooth muscle cells were plated onto collagen I-coated Flex I culture plates and subjected to 25% static stretch on day 4 of culture. Static stretch induced an increase in the expression of c-fos, fosB, fra-1, c-jun, and junB. The expression of both c-fos and junB was maximally induced at 30 min by static stretch. The peak induction for fosB and c-jun occurred at 1 h, whereas the peak of fra-1 induction occurred between 1 and 2 h after application of stretch. Treatment of myometrial cells with progesterone (100 nM, 400 nM, 1 microM) for 1 or 6 h before the application of static stretch did not affect the magnitude of the c-fos response. However, 24 h of progesterone exposure reduced the magnitude of c-fos and fosB stretch induction at both the 400 nM and 1 microM doses. These data indicate that several members of the AP-1 family are stretch-responsive genes in myometrial smooth muscle cells. This response can be attenuated by pretreatment with progesterone; however, the requirement for longer pretreatment times suggests that the inhibitory actions of progesterone do not occur through a direct action of the progesterone receptor within the promoter regions of AP-1 genes.     

A critical role for elastin signaling in vascular morphogenesis and disease

Vascular proliferative diseases such as atherosclerosis and coronary restenosis are leading causes of morbidity and mortality in developed nations. Common features associated with these heterogeneous disorders involve phenotypic modulation and subsequent abnormal proliferation and migration of vascular smooth muscle cells into the arterial lumen, leading to neointimal formation and vascular stenosis. This fibrocellular response has largely been attributed to the release of multiple cytokines and growth factors by inflammatory cells. Previously, we demonstrated that the disruption of the elastin matrix leads to defective arterial morphogenesis. Here, we propose that elastin is a potent autocrine regulator of vascular smooth muscle cell activity and that this regulation is important for preventing fibrocellular pathology. Using vascular smooth muscle cells from mice lacking elastin (Eln(-/-)), we show that elastin induces actin stress fiber organization, inhibits proliferation, regulates migration and signals via a non-integrin, heterotrimeric G-protein-coupled pathway. In a porcine coronary model of restenosis, the therapeutic delivery of exogenous elastin to injured vessels in vivo significantly reduces neointimal formation. These findings indicate that elastin stabilizes the arterial structure by inducing a quiescent contractile state in vascular smooth muscle cells. Together, this work demonstrates that signaling pathways crucial for arterial morphogenesis can play an important role in the pathogenesis and treatment of vascular disease.     

Actin isoform and α1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch

Summary The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and α_1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of α_1B -adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in α_1B-adrenoceptor or β/γ-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.     

Rapid effects of stretched myocardial and vascular cells on gene expression of neonatal rat cardiomyocytes with emphasis on autocrine and paracrine mechanisms

Passive stretch of the heart has a direct effect on cardiomyocytes and other cell types including cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells (VSMCs). Cardiomyocytes are targets for the action of peptide growth factors found in myocardium, suggesting an autocrine or paracrine model of the hypertrophic process. In this study we examined stretch-dependent cellular communication between cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. Stationary cardiomyocytes were incubated with stretch-conditioned medium (CM0-CM60) derived from stretched (for 0-60 min) cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. The expression levels of protooncogenes (as c-fos, c-jun, and fra-1) were measured, and as an indication of a hypertrophic response the expression of atrial natriuretic peptide (ANP) was measured. Stationary cardiomyocytes that have been incubated for 30 min with CM from stretched (for 0-60 min) cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs showed distinct gene expression patterns that were time-dependent and cell-type specific. In stationary cardiomyocytes, CM derived from stretched cardiomyocytes caused decreased c-fos and fra-1 expression by 37 and 20%, respectively (CM30), elevated c-jun expression by 20% (CM45-CM60), and increased ANP expression by 106% (CM45). CM derived from stretched cardiac fibroblasts caused increased c-fos expression by 41% (CM60), no significant changes in c-jun expression, and increased fra-1 and ANP expression by 39 and 20%, respectively (CM45). CM derived from stretched VSMCs induced an initial decrease in c-fos expression followed by an increase of 13% (CM45) and induced increased c-jun, fra-1, and ANP expression by 39, 24, and 22%, respectively. CM15-CM60 derived from stretched endothelial cells caused decreased c-fos, c-jun and fra-1 expression by 20, 25, and 25%, respectively, and increased ANP expression by 18%. Our data indicate that gene expression of cardiomyocytes in stretched myocardium is regulated by mediators released by cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. This observation emphasizes the involvement of nonmyocyte cells in the early stages of cardiomyocyte hypertrophy caused by cardiac stretch.     

Integrins in mechanotransduction

Mechanical forces are crucial to the regulation of cell and tissue morphology and function. At the cellular level, forces influence cytoskeletal organization, gene expression, proliferation, and survival. Integrin-mediated adhesions are intrinsically mechanosensitive and a large body of data implicates integrins in sensing mechanical forces. We review the relationship between integrins and mechanical forces, the role of integrins in cellular responses to stretch and fluid flow, and propose that some of these events are mechanistically related.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.     

Self-assembly of elastin-based peptides into the ECM: the importance of integrins and the elastin binding protein in elastic fiber assembly

The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.     

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.

We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.     

Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia.     

Cyclic stretch enhances reorientation and differentiation of 3-D culture model of human airway smooth muscle

Activation of airway smooth muscle (ASM) cells plays a central role in the pathophysiology of asthma. Because ASM is an important therapeutic target in asthma, it is beneficial to develop bioengineered ASM models available for assessing physiological and biophysical properties of ASM cells. In the physiological condition in vivo, ASM cells are surrounded by extracellular matrix (ECM) and exposed to mechanical stresses such as cyclic stretch. We utilized a 3-D culture model of human ASM cells embedded in type-I collagen gel. We further examined the effects of cyclic mechanical stretch, which mimics tidal breathing, on cell orientation and expression of contractile proteins of ASM cells within the 3-D gel. ASM cells in type-I collagen exhibited a tissue-like structure with actin stress fiber formation and intracellular Ca²⁺ mobilization in response to methacholine. Uniaxial cyclic stretching enhanced alignment of nuclei and actin stress fibers of ASM cells. Moreover, expression of mRNAs for contractile proteins such as α-smooth muscle actin, calponin, myosin heavy chain 11, and transgelin of stretched ASM cells was significantly higher than that under the static condition. Our findings suggest that mechanical force and interaction with ECM affects development of the ASM tissue-like construct and differentiation to the contractile phenotype in a 3-D culture model.     

Responses of vascular smooth muscle cell to extracellular matrix degradation.

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.     

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.     

Strain history and TGF-β1 induce urinary bladder wall smooth muscle remodeling and elastogenesis

Mechanical cues that trigger pathological remodeling in smooth muscle tissues remain largely unknown and are thought to be pivotal triggers for strain-induced remodeling. Thus, an understanding of the effects mechanical stimulation is important to elucidate underlying mechanisms of disease states and in the development of methods for smooth muscle tissue regeneration. For example, the urinary bladder wall (UBW) adaptation to spinal cord injury (SCI) includes extensive hypertrophy as well as increased collagen and elastin, all of which profoundly alter its mechanical response. In addition, the pro-fibrotic growth factor TGF-β1 is upregulated in pathologies of other smooth muscle tissues and may contribute to pathological remodeling outcomes. In the present study, we utilized an ex vivo organ culture system to investigate the response of UBW tissue under various strain-based mechanical stimuli and exogenous TGF-β1 to assess extracellular matrix (ECM) synthesis, mechanical responses, and bladder smooth muscle cell (BSMC) phenotype. Results indicated that a 0.5-Hz strain frequency triangular waveform stimulation at 15% strain resulted in fibrillar elastin production, collagen turnover, and a more compliant ECM. Further, this stretch regime induced changes in cell phenotype while the addition of TGF-β1 altered this phenotype. This phenotypic shift was further confirmed by passive strip biomechanical testing, whereby the bladder groups treated with TGF-β1 were more compliant than all other groups. TGF-β1 increased soluble collagen production in the cultured bladders. Overall, the 0.5-Hz strain-induced remodeling caused increased compliance due to elastogenesis, similar to that seen in early SCI bladders. Thus, organ culture of bladder strips can be used as an experimental model to examine ECM remodeling and cellular phenotypic shift and potentially elucidate BMSCs ability to produce fibrillar elastin using mechanical stretch either alone or in combination with growth factors.     

Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions

We studied the effect of cyclic mechanical stretching on the proliferation and collagen mRNA expression and protein production of human patellar tendon fibroblasts under serum-free conditions. The role of transforming growth factor-beta1 (TGF-beta1) in collagen production by cyclically stretched tendon fibroblasts was also investigated. The tendon fibroblasts were grown in microgrooved silicone dishes, where the cells were highly elongated and aligned with the microgrooves. Cyclic uniaxial stretching with constant frequency and duration (0.5 Hz, 4 h) but varying magnitude of stretch (no stretch, 4%, and 8%) was applied to the silicone dishes. Following the period of stretching, the cells were rested for 20 h in stretching-conditioned medium to allow for cell proliferation. In separate experiments, the cells were stretched for 4h and then rested for another 4 h. Samples of the medium, total cellular RNA and protein were used for analysis of collagen and TGF-beta1 gene expression and production. It was found that there was a slight increase in fibroblast proliferation at 4% and 8% stretch, compared to that of non-stretched fibroblasts, where at 8% stretch the increase was significant. It was also found that the gene expression and protein production of collagen type I and TGF-beta1 increased in a stretching-magnitude-dependent manner. And, levels of collagen type III were not changed, despite gene expression levels of the protein being slightly increased. Furthermore, the exogenous addition of anti-TGF-beta1 antibody eliminated the increase in collagen type I production under cyclic uniaxial stretching conditions. The results suggest that mechanical stretching can modulate proliferation of human tendon fibroblasts in the absence of serum and increase the cellular production of collagen type I, which is at least in part mediated by TGF-beta1.     

Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells

Angiotensin II (AII) binds to G protein-coupled receptor AT(1) and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α(1)-, α(5)-, α(V)-, and β(1)-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α(5)β(1)-integrin, and by ～60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α(1)β(1). Furthermore, neutralizing antibody against β(1)-integrin and silencing of α(1), α(5), and β(1) expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α(5)β(1) and α(1)β(1)) in AII-induced proliferation of VSMC.     

Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation.Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0–10 ng/ml transforming growth factor (TGF)β₁. mRNA expression was analyzed by quantitative real-time PCR.New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ₁ strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen.The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.     

Modulation of vascular smooth muscle cells proteoglycan synthesis by the extracellular matrix

In this study, we investigated the effect of the extracellular matrix (ECM) secreted by vascular cells on proteoglycan (PG) synthesis by vascular smooth muscle cells in culture. PG synthesis of human aortic smooth muscle cells plated on plastic or the matrices derived from vascular endothelial cells, vascular smooth muscle cells, or THP-1 macrophages was characterized. Smooth muscle cell and macrophage matrices increased both secreted and cellular smooth muscle cells PG production by 2.5-fold to 3.9-fold, respectively, over plastic and endothelial cell matrix. Macrophage matrix was more potent than smooth muscle cell matrix in this regard. Selective enzymatic removal of chondroitin sulfates, collagen, and elastin from smooth muscle cell matrix enhanced the stimulation of PG synthesis, as did the removal of chondroitin sulfates from macrophage matrix. PG turnover rates were similar for smooth muscle cells plated on the three matrices. The newly synthesized PG from cultures plated on smooth muscle cell-, and macrophage-derived matrices had greater charge density, larger molecular size, and longer glycosaminoglycan chains than those from endothelial cell matrix cultures. These data show that the ECM plays a major role in modulating vascular smooth muscle cell PG metabolism in vitro.