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Call For Papers: Translational Physiology
Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, New York
ABSTRACT
Studies were performed to examine the extent to which mechanical stimuli mediate control of angiogenesis in bladder cells both in vitro and in vivo. Differential gene expression between control nonstretched and cyclically stretched bladder smooth muscle cells was assessed using oligonucleotide microarrays and pathway analysis by the web tool Fast Assignment and Transference of Information (FatiGO). Data showed that a substantial proportion (33 of 86) of mechanically responsive genes were angiogenesis-related and include cytokines, growth-related factors, adhesion proteins, and matricellular, signal transduction, extracellular matrix (ECM), and inflammatory molecules. Integrative knowledge of protein-protein interactions revealed that 12 mechano-sensitive gene-encoded proteins have interacting partner(s) in the vascular system confirming their potential role in paracrine regulation of angiogenesis. Angiogenic genes include matricellular proteins such as Cyr61/CCN1, CTGF/CCN2 and tenascin C, components of the VEGF and IGF systems, ECM proteins such as type I collagen and proteoglycans, and matrix metalloproteinases. In an in vivo model of bladder overdistension, 5 of 11 mechano-responsive angiogenic genes, independently tested by real-time PCR, were upregulated as a result of pressure overload including Cyr61/CCN1, CTGF/CCN2, MCP-1, VEGF-A, MMP-1, and midkine. Meanwhile, the molecular anatomy of angiogenic gene promoters reveals the presence of GA box-binding for the myc-associated zinc finger protein, MAZ, often found adjacent to binding sites for mechano-responsive transcription factors (e.g., NF-
B), suggesting that the coordinated activity of these factors may induce selective angiogenic gene transcription. These data suggest that mechanical control of angiogenic genes is an integral part of the adaptive and plasticity responses to mechanical overload.
mechanical stretch; bladder obstruction; angiogenesis; differential gene expression; extracellular matrix
MECHANICAL FORCES PLAY an important role in regulating cell biochemistry, fate, and behavior. Muscle contraction, which derives from internal stresses, begins in utero and imposes both a local cellular stress and an overall tissue deformation throughout muscular organs. Overall, mechanical cues, whether internally generated or externally imposed, orchestrate the differentiation and growth of most distensible organ systems during development and adult life, initial patterning and early embryonic morphogenesis being strictly controlled by intrinsic genetic factors and the local chemical environment. The urinary bladder, in particular undergoes tonic distension and periodic changes in volume, both in utero and after birth. In human embryogenesis, bladder cycling/volume work begins as early as the fifth month of gestation, which marks the onset of an accelerated increase of bladder growth and capacity (6, 7). In the Wnt-4 knockout mouse, which is characterized by lack of kidney formation, the bladder develops but does not perform any volume work since it does not cycle urine (60). In these mice, the bladder wall appeared almost acellular but thick and fibrotic compared with the urodynamically active hyperplastic bladder of wild-type mice. Reversibly, if the volume work of the bladder intensifies as a result of anatomical or functional obstruction, profound tissue remodeling occurs and translates into hypertrophic and fibrotic responses (36). These adaptive responses enable the bladder to compensate transiently for the extra effort required for the voiding process, but ultimately they result in altered detrusor muscle contractility and bladder wall compliance, physiological insufficiency, and organ failure.
The urinary bladder is a structure thin enough to survive on diffused nutrition until a blood supply is established. However, a satisfactory vascularization remains an important restraint on its adaptive capability to mechanically challenging conditions, i.e., urethral obstruction. Indeed, the structure and mechanical performances of the bladder are extremely sensitive to overdistension-induced ischemia/hypoxia. Several studies have demonstrated that altered contractility of the bladder as a result of sustained urethral obstruction was associated with hypoxia/ischemia and a significant decrease of blood flow to the mucosa and detrusor smooth muscle layers (28, 45). Similarly, detrusor overactivity of obstructed bladder paralleled intermittent reduction of blood flow and local hypoxia (33). In the experimental rabbit model of urinary obstruction, a decrease of blood supply as a result of overdistension-induced hypoxia coupled with impaired angiogenesis in the detrusor muscle marked a shift to a decompensated myopathy (40). Conversely, rats were more resistant than rabbits to the onset of bladder decompensation, presumably owing to a sustained active angiogenesis that rehabilitates bladder blood flow (16). However, there is a paucity of molecular data in the literature to support these observations essentially because the importance of angiogenesis in bladder response to overdistension/hypertrophy has received little attention compared with other neurogenic and genetic factors. Interestingly, the paradigm of mechanical overload relationship to angiogenesis has been better demonstrated in the cardiovascular system. Studies have shown that cardiomyocyte hypertrophy due to mechanical overload increases diffusion distance, resulting in reduced oxygen supply/ischemia in the myocardium (30, 51). The resulting neovascularization/cardiac angiogenesis further contributes to the development of cardiac hypertrophy, and its impairment induces heart failure. The expression of a pool of angiogenic and vasculogenic factors has been associated with cardiac overload-induced hypertrophy (23). Whether a similar process takes place in a mechanically overloaded smooth muscle-rich organ such as the obstructed bladder is unknown. Superficial similarities in the response of the heart and bladder to mechanical stimuli suggest that similar molecular mechanisms may be involved.
Angiogenesis refers to the growth of new blood vessels from pre-existing ones and involves the activation of quiescent endothelial cells, which start to migrate, proliferate, and organize themselves into tubular structures. This is a highly dynamic process that requires complex interactions of extracellular matrix (ECM) molecules, proteases, soluble mediators, and various resident cells (24). In overly distended obstructed bladders, the main molecular alterations include those of contractile proteins expression and/or regulation, increases in extracellular collagen remodeling, reorientation of smooth muscle cells, and alterations in myofilament length and orientation within the cells (36, 38). These changes are consistent with the response of a highly physically stressed tissue undergoing repair. Our laboratory has previously reported that the cysteine-rich protein 61 (Cyr61/CCN1), a novel proangiogenic molecule, is upregulated from the early stages of obstruction and may play an important role in angiogenic remodeling (10, 13). However, information about the impact of mechanical strain on the overall gene program of angiogenesis in the bladder remains scant. We postulate that the expression of angiogenic genes, perhaps of equal importance to remodeling and cytoskeletal genes in the mechanical response of bladder smooth muscle cells, is modulated as well. To test this hypothesis, we used microarray analysis to address, on a large-scale basis, the genetic changes resulting from the application of mechanical forces to cultured bladder smooth muscle cells. A greater emphasis was placed on the subset of genes involved in the paracrine regulation of angiogenesis in both mechanically stimulated bladder smooth muscle cell cultures and an animal model of bladder outlet obstruction.
MATERIALS AND METHODS
Cell culture.
Primary culture of human bladder smooth muscle cells (HBSMCs) were obtained from Cambrex/Lonza (Allendale, NJ). Cells were cultured in SmGM-2 containing 5% fetal bovine serum (FBS) and a supplement of growth factors and cytokines packaged in the SingleQuots Kit (Cambrex). HBSMCs maintain differentiated properties in culture after four to five passages.
Mechanical stimulation of HBSMCs.
A timetable for the experimental conditions used is shown in Table 1. In brief, cells were plated on six-well silicone elastomer-bottomed culture Flex plates coated with type I collagen and incubated for 24 h. Medium was then replaced with SmGM-2 containing 0.5% FBS and a 1/10th of the concentration of growth factor supplement, and cells were incubated for an additional 24 h. The presence of a minimum concentration of growth factor supplement is necessary for survival of HBSMCs. The medium was then removed and replaced by a fresh one. For experimental plates, cyclic stretch was applied to the cells using the FX-4000 Flexercell Tension Plus System (Bioflex; Flexcell, Hillsborough, NC) as described previously (14). Cells were subjected to a maximum of 15% strain magnitude at a frequency of 0.3 Hz for either 1 h (t1) or 24 h (t24). This stretch regimen produced optimal conditions for imparting mechanical stimulation without inducing cell injury. For control, cells were kept under static conditions in the same culture medium. After completion of either the stretch regimen or incubation time period under static conditions, cells were pooled from the six wells of each Flex plate and processed for RNA analysis. Pooling of the content of at least six individual wells was necessary to extract sufficient amount of RNA for analysis. Stretch and control experiments, using the same pool of cells, were carried out simultaneously and analyzed identically.
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39,000 well-substantiated human genes. Arrays were washed at 25°C with 6x SSPE (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA, and 0.01% Tween 20) followed by a stringent wash at 50°C with 100 mM MES and 0.01% Tween 20. The arrays were then stained with phycoerythrein-conjugated streptavidin (Molecular Probes, Eugene, OR), and the fluorescence intensities were determined using a laser confocal scanner (Hewlett-Packard, Palo Alto, CA). The scanned images were analyzed using Microarray Suite 5.0 software (Affymetrix), following the user guidelines. The signal intensity for each gene was calculated as the average intensity difference, represented by [
(PM–MM)/(number of probe pairs)], where PM and MM denote perfect-match and mismatch probes, respectively. The annotation for each probe set of the U13 array was verified by downloading the latest annotation files from Affymetrix. Transcripts expressed in HBSMCs under static conditions or under stretched conditions for 1 and 24 h were compared. We used a 2 x 2 comparison of gene expression levels (depicted as intensity levels on the microarrays) from two separate experiments, i.e., two chips were used on different RNA preparations. Fold changes were calculated separately as the ratio of normalized average intensity difference (test sample) divided by normalized average intensity difference (baseline sample). Thresholds were set for fold change (twofold and greater either increase or decrease) and absolute difference between normalized average intensity differences as provided by the Affymetrix analysis. The online Fast Assignment and Transference of Information (FatiGO) web tools were used for pathway analysis (3, 4). FatiGO and FatiGO Plus, which are accessible online at http://www.fatigo.org, perform functional enrichment analysis by comparing two lists of genes by means of a Fisher's exact test. Gene modules used in the test are defined in different ways, which, in our study, include functional criteria (gene ontology) or regulatory criteria (e.g., transcription factor targets). Information on unknown mechano-responsive genes was collected using GeneCards, an integrated database of human genes that includes automatically mined genomic, proteomic and transcriptomic information, and gene expression and function (8). GeneCards is accessible online at http://www.genecards.org.
Quantitative real-time PCR analysis of mRNA.
Real-time PCR was performed on aliquots of cDNA reverse-transcribed from total RNA as previously described (41). The reaction was performed with cDNA equivalent to 20 ng of RNA, 300 nM each of forward and reverse primers, and Syber Green real-time PCR Mix (Superarray) according to the manufacturer's protocol in the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Highly specific primers were designed using the Web-based primer design program Primer3 and obtained commercially (Invitrogen). The cycling parameters were: AmpliTaq activation 95°C for 10 min, denaturation 95°C for 15 s, and annealing/extension 60°C for 1 min (40 cycles). Control and experimental cDNA, cDNA for the derivation of standard curves, and no-template controls were run in triplicate. A dissociation curve was obtained at the end of each reaction to verify the presence of a single product with the appropriate melting point temperature for each product. Triplicate CT values were analyzed with Microsoft Excel using the comparative CT (hhCT) method. The amount of transcripts (2–hhCT) was obtained by normalizing to an endogenous reference (18S rRNA) relative to a calibrator (one experimental sample).
Preparation of nuclear extracts.
Nuclear extracts were prepared from isolated nuclei from control and mechanically stretched cells as previously described (35). Briefly, cells were placed in hypotonic buffer [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol (DTT)] and incubated on ice for 15 min. Igepal CA-630 was added to a final concentration of 0.6%, and the mixture was vortexed vigorously for 10 s. Nuclei were recovered by centrifugation at 3,300 g for 30 s at 4°C and extracted in buffer containing 20 mM HEPES pH 7.9, 0.42 M NaCl, 25% glycerol, 1.5 mM MgC12, 0.2 mM ethylenediamine tetraacetic acid (EDTA), and 0.5 mM DTT for 30 min at 4°C by gentle shaking. The extract was centrifuged for 15 min at 25,000 g, and the supernatant was then frozen at –70°C. All buffers contained a protease inhibitor cocktail [2 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 1.4 pM trans-epoxysuccinyl-L-leucylamido(4-guanidinobutane), 1 µM leupeptin, and 0.3 pM aprotinin (Roche Bioscience)]. Protein concentration was determined using the Bradford assay.
Transcription factor/DNA array.
Transcription factor activity in nuclear extracts was analyzed using the TranSignal Protein/DNA array I (Panomics, Redwood City, CA) according to the manufacturer's specifications. This assay enables the simultaneous detection and semiquantitative comparison of the DNA-binding activity of up to 54 different transcription factors from nuclear extracts from cells treated under two different conditions. In brief, biotin-labeled DNA-binding oligonucleotides (TranSignal Probe Mix) were incubated with 10 µg of nuclear extract at 15°C for 30 min to allow the formation of protein/DNA complexes. The complexes were separated from the free probes by 2% agarose gel electrophoresis in 0.5x Tris-borate-EDTA at 120 V for 15 min. The probes in the complexes were then extracted, ethanol precipitated, and hybridized to the TranSignal Protein/DNA Array. Detection of signals was obtained using an enhanced chemiluminescence imaging system.
Experimental model of bladder overdistension.
Animal studies were reviewed and approved by the Institutional Review Board of the State University of New York Downstate Medical Center. Outlet obstruction of the bladder was surgically induced and maintained for 7 and 14 days. The bladder responds to increased after-load as a result of partial obstruction by undergoing hypertrophy/hyperplasia to normalize wall tension. Hypertrophy/hyperplasia is associated eventually with bladder overactivity and can be well appreciated in the 7-day partially obstructed rat bladder as shown in our previous studies (13, 14). Persistence of the after-load (up to 14 days) leads eventually to a decompensated myopathy, which commonly occurs after severe/total obstruction but rarely in partially obstructed bladder of rats. (56). Thus, the molecular changes are ideally appreciated after 7 and 14 days postobstruction. Experimentally, outlet obstruction of the bladder was induced in Sprague-Dawley female rats (Charles River Laboratories, Wilmington, MA) through partial obstruction of the urethra as previously described (14). Briefly, a 2-0 silk ligature was tied loosely around the urethra so that the lumen diameter was constrained to 1 mm. In the sham control, the bladder and urethra were exposed, but no ligature was applied. At the end of the period of obstruction, the bladder was excised rapidly and processed for molecular and histochemical analyses. For molecular studies, total RNA was extracted using RNAeasy protocol (Qiagen) and processed for measurement of specific transcript levels by real-time PCR as described above. Capillary density within the bladder wall was examined by histochemistry. Unfixed flat-mounted bladders were washed with PBS and incubated for 24 h in serum-free medium containing 10 µg/ Ulex europaeus agglutinin (UEA)-1 (Vector Biolab), a fluorescently labeled plant lectin commonly used as a marker of vessels and capillaries. Flat mounts were then analyzed by fluorescence microscopy. Capillary density was quantified by summing capillary junctions within four equal areas, each 0.16 mm2, and was expressed as the number of junctions per 0.64 mm2.
Statistical analysis.
Data were expressed as means ± SE. To test differences among several means for significance, a one-way ANOVA with the Newman-Keuls multiple-comparison test was used. Post hoc unpaired t-test was used to compare two means/groups, and P values <0.05 or <0.01 were considered significant.
RESULTS
Overall gene expression profile after short- and long-term stretch.
We used the Human Genome U133 Plus 2.0 Array from Affymetrix to perform a large-scale analysis of mechano-responsive genes in HBSMCs. The cells were mechanically stimulated for shorter (1 h) and longer (24 h) periods of time. Comparisons were made with cells cultured under static conditions. Based on the default parameters of the Affymetrix Genechip analysis software, 33% of genes spotted on the array were scored as being present (or marginal) in the cells under static and mechanically stimulation conditions. The normalization factors used to compare the hybridization data from the RNA samples were equivalent. After 1 h exposure to mechanical stretch, only 85 genes had a significantly altered expression levels by more than twofold. None of the differentially expressed genes exhibited altered mRNA levels by more than fivefold. Of these, 30 were upregulated and 59 genes were downregulated by the mechanical stimulus. When the cells were exposed to the mechanical stimulus for 24 h, 86 genes were differentially regulated. The transcript levels of 27 genes decreased, while the expression of 59 genes was increased by the mechanical stimulus.
Functional annotation was conducted using the web tool FatiGO to categorize differentially expressed genes based on their biological process or molecular functions (3). Figure 1 shows that a large number of differentially expressed genes were involved in signal transduction and regulation of metabolic processes, response to wound healing, and cell-cell signaling. After 1 h of mechanical stimulation, higher numbers of genes were involved in embryonic development and epigenic regulation of gene expression, while the majority of differentially expressed genes after 24 h of stretching was involved in cell-cell signaling, chemokine production, cell cycle regulation, and cell motility.
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To further demonstrate the selective activation by mechanical strain of genes involved the paracrine signaling of angiogenesis, we examined whether proteins encoded by mechano-responsive genes and secreted in the extracellular milieu have interacting partners in the vascular system e.g., endothelial cell, vascular smooth muscle cells, and pericytes. We took advantage of recent advances in the availability of protein-protein interaction databases that have been constructed from diverse organisms using yeast-two-hybrid, tag/pull-down, and literature search approaches. We generated a protein-protein interaction network in which all known interacting partners of the initially selected molecules were determined and incorporated into the model. Our data showed that 12 mechano-sensitive gene-encoded proteins have interacting partner(s) in the vascular system (Fig. 2). For instance, the matricellular proteins Cyr61 and CTGF support adhesion and proliferation of endothelial cells via interaction with 
vβ3 and 5β1 integrins and contactin-1, which itself was shown to be mechano-sensitive and promote endothelial and vascular cell growth (1, 15, 18). Similarly, several components of the IGF system interact with vascular partners that drive the angiogenic process. In addition, IGF-I is also known to stimulate the production of VEGF and promote endothelial and vascular smooth muscle cell proliferation (54). IGFBP-3 positively and independently of IGF-I regulates blood vessel growth through unknown mechanisms (32), while IGFBP-2 and IGFBP-4 have been shown to exhibit antiangiogenic properties (21, 29). Clearly mechanical stretch modulates the expression of a broad range of angiogenic and antiangiogenic molecules.
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B, and Egr-1, which we have previously reported as stretch-sensitive genes (12, 35). Other active transcription factors include CREB, Ets, MEF-1, AP-2, USF-1, and VDR. A stronger DNA binding activity for Smad3/4 were detected in nuclear extracts from stretched versus control cells.
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B, and Pax-3 are the most abundant in both proximal (<1,000 bp) and distal (<5,000 bp) 5' regions of the analyzed genes (Table 5). MAZ, which refers to myc-associated zinc finger protein, is a constitutively expressed GA box-binding transcription factor targeting a large number of angiogenic genes including MMP-28, CCL19, midkine, laminin β3 and β1, ECGF1, IGFBP-2, integrin v, integrin 7, IGFBP-3, COL3A1, IGFBP-4, COL1A1, spondin 1, CSPG 4, and thrombospondin 1.
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To optimize their load-bearing potential, mechanically challenged smooth muscle cells of the bladder elicit adaptive responses acutely to rapidly alter cell function and chronically by retooling their cytoskeletal structures and remodeling the extracellular environment. Initially, mechanical signals are transmitted through selective transduction pathways via transmembrane receptors (e.g., ion channels, G protein-coupled receptors, integrins) that are physically connected to the actin cytoskeleton. Cytoskeletal proteins provide a continuous and dynamic link between virtually all cellular structures, and thus, they enable internal structures such as chromatin to respond directly and immediately to mechanical forces. These mechano-sensing pathways are closely integrated with the concerted expression of specific groups of genes.
Remarkably, the expression of a fraction of genes (<90 out of >39,000) covered by the Affymetrix U133 Plus chip was altered by mechanical stretch above a twofold threshold. This highly selective modulation of gene expression is reflected also in Adam et al.'s study (2) that showed that only 20 of 11,731 gene oligonucleotides spotted on the HG-U133A array were mechano-sensitive in HBSMCs. The list of differentially expressed genes commonly described in this study and that of Adam et al. included those for LIF, BMP-2, and tenascin C. In addition, genes of the "fetal gene program" such as IGF-I, Cyr61, and CTGF, which we have previously identified as mechano-sensitive genes in bovine bladder smooth muscle cells by using single gene-based analytical techniques (11, 14), were modulated by mechanical stretch, which corroborates results of the microarray analysis approach. Of note is that stretch stimulation of human and bovine bladder smooth muscle cells did not lead to a comparable degree of induction of fetal genes (i.e., magnitude of gene induction was lower in human vs. bovine cells), perhaps because cells from different species were not initiated and propagated with the same media and under identical culture conditions. Additionally, our list of differentially expressed genes included those encoding IGF-I and bFGF, although the latter did not attain the twofold change cutoff (data not shown), but our list did not include genes encoding antiapoptotic factors such as cIAP and Hsp-70. These data are in disagreement with Galvin et al.'s study (25), which showed increased protein levels for not only IGF-I and bFGF, but also cIAP-1 and Hsp-70 in HBSMCs subjected to cyclic stretch. However, although changes in mRNA abundance for most genes reflect those at the protein level as well; it remains to be determined whether cIAP and Hsp-70 gene expression is transcriptionally or posttranscriptionally controlled in mechanically stimulated cells. Differences between results from various studies may otherwise be due to cell culture conditions and/or the stretch regimen used.
Our data showed that cyclic stretch induced the rapid and transient expression of a number of IE responsive genes, many of which encode regulatory molecules typified by TF and cofactors (e.g., Egr-1, SRF), ECM-associated proteins (e.g., MCP-1, Cyr61, CTGF), signaling molecules (e.g., Gene 33), and cytoskeletal proteins. In addition, new IE responsive genes such as MRTF-B and Fzd 3 were identified in mechanically stimulated cells as well. In particular, MRTF-B also known as MLK2/MAL16, is a transcriptional coactivator that promotes SRF-dependent transcription in response to intracellular cytoskeletal changes, e.g., Rho GTPase activation and actin signaling (52). MRTF-B gene deficiency resulted in defective smooth muscle gene expression in several smooth muscle-rich organs, which contributed to a lethal phenotype in mice (48). Thus, even though the IE gene response to mechanical stretch is rapid, transient, and stereotypic, it also is an integral part of the adaptive and plasticity response.
The general tenet of the IE gene response is to alter the intracellular signaling in stimulated cells and transmit the mechanical signal to neighboring cells via soluble factors. In this context, we found that a little more than one-third of the identified mechano-responsive genes encode secreted proteins involved in the paracrine regulation of angiogenesis. Among the most notable angiogenic genes are those of the VEGF and IGF systems, ECM proteins such as fibrillar and basement membrane collagens that regulate endothelial cell migration and capillary bifurcation, ECM-associated proteins and matricellular proteins that affect nearly all aspects of vessel sprouting and sheathing by smooth muscle and smooth muscle-like cells (e.g., pericytes). Complementing this valuable information generated through oligonucleotide array with integrative knowledge of protein-protein interactions enabled us to confirm the putative paracrine regulation of angiogenesis by many of the secreted mechano-sensitive gene-encoded proteins. Essentially, we found that 1) most secreted soluble and ECM factors have known interacting partners on vascular cells, and 2), mechanical stretch induced the expression of many vascular growth factor genes but not their respective receptors, which supports the concept that most soluble factors act in a paracrine fashion. VEGF-A, the best characterized and the most studied of the VEGF family members, is induced by IE gene-encoded proteins such as Cyr61, and therefore, VEGF-A may not only be the primary target of mechanical stretch but also a secondary target of IE gene expression (69). VEGF-D, which has structural similarity and shares less homology with VEGF-A, was stretch sensitive as well (50). VEGF-A and -D interact with transmembrane tyrosine kinase receptors, e.g., VEGF-R1 and VEGF-R2, which are selectively expressed on vascular endothelial cells. Neither VEGF-R1 nor VEGF-R2 gene was responsive to stretch in HBSMCs even though their ligands encoded by the VEGF genes were upregulated. However, other studies reported that VEGF-R2 gene expression was increased in mechanically stimulated endothelial cells, suggesting that cells respond in a unique but complementary fashion to mechanical stimuli (68).
In addition to soluble growth factors, mechanical strain activated the expression of proangiogenic ECM protein genes encoding laminin B1, type I and type IV collagens, and tenascin C and repressed the expression of antiangiogenic ECM constituents such as thrombospondin 1 and spondin. ECM molecules play a pivotal role in angiogenesis (55). Overall, ECM proteins drive capillary morphogenesis through sustained signaling that maintains cytoskeletal organization and shape of endothelial cells (20). They also immobilize and organize gradients of angiogenic cytokines and growth factors, which are required for vascular cell adhesion, migration, and proliferation. During neoangiogenesis, new vessels sprout out of existing vessels and grow along the growth factor gradient. To mediate this outgrowth, endothelial cells have to degrade the basement membrane and the surrounding ECM with the help of proteases, mainly metalloproteases, serine, and cysteine proteases. In this context, we found that both MMP-1 and MMP-28 transcript levels were increased by mechanical stretch. Increased MMP activity was previously shown to mediate, at least in part, VEGF-A-induced angiogenesis in vivo (22). MMP-1, in particular, is required for invasion into the interstitial space of endothelial cells (62). MMP-28 also known as epilysin, the latest addition to the MMP family, has putative functions in tissue repair and angiogenesis (43). However, tissue inhibitors of metalloproteinase (TIMP), the endogenous inhibitors of MMPs, have also been reported to be activated by mechanical stretch (49). Thus, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. This coactivation of both pro- and antiangiogenic factors can be viewed as a balanced system that both forms new blood vessels and prevents vessel overgrowth.
Meanwhile, recent studies have described the ECM-associated proteins Cyr61 and IGFBP-3 as soluble progenitor cell-active factors that promote angiogenesis in part through mobilization and recruitment of endothelial progenitor cells (9, 58). Preliminary data from our laboratory have shown that Cyr61 has the potential to induce adhesion, migration, and differentiation of CD34+ hematopoietic stem cells into endothelial progenitor cells (EPCs), suggesting its potential role in vasculogenesis (Liu H, Amir J, and Chaqour B. Unpublished observations). Consistent with this, ablation of the Cyr61 gene in mice resulted in embryonic lethality due to defects in the formation and bifurcation of new capillaries, suggesting that Cyr61 is required for proper vasculogenesis (44). Similarly, IGFBP-3 acts as chemoattractant for cultured EPCs and promoted vascular regrowth in vivo (9). Whether the expression of these proteins exerts vasculogenic and vasculotriophic effects in the bladder wall during the early phases of the obstruction is unknown and remains to be investigated. It is now well established that a combination of both vasculogenesis, de novo vessel formation from hemangioblasts, and angiogenesis, budding from preexisting blood vessels, takes place in a large number of diseases involving remodeling of the vasculature (5).
The elucidation of the molecular mechanisms governing the activation of proangiogenic phenotype is central for understanding and controlling the cell's response to mechanical stimuli. One interpretation of how selective gene regulation of angiogenic genes is achieved by the mechanical stimulus is that functionally-related genes might share common transcriptional regulators. Since the binding activities of transcription factors often depends on posttranscriptional modifications that are not detectable at the RNA levels, we used a protein/DNA array to profile the DNA binding activity of multiple TF. We identified numerous TF, which bind to "mechano-responsive" promoter elements in mechano-sensitive genes. The majority of proangiogenic genes contains MAZ, GATA-4, NF-B, and NKx-2.5 binding sites within their core promoter. MAZ binds to the same cis-element in the promoters of the genes for the receptor for serotonin 1A, endothelial nitric-oxide synthase and the receptor for parathyroid hormone (59). In our analysis, MAZ consensus sites were found mostly in ECM proteins, MMPs, and integrin promoters. The molecular anatomy of the promoter region of these genes also revealed the presence of GA-rich motifs for MAZ adjacent to binding sites for other TF (e.g., NF-B, GATA-4), suggesting that the combined action of these factors modulates transcription of functionally related genes. For instance, a physical and/or functional interaction between MAZ and NF-B has been documented (53). The emerging theme in mechanical control of gene expression is that regulation results from functional interactions between and among several TF.
Another interesting result of our study design is correlation of the in vitro data to in vivo conditions. Indeed, the in vitro biomechanical system used in our study involves overdistension of the elastomeric membrane of the wells subjecting the adherent cells to a reproducible degree of mechanical deformation. While this system facilitates study of the proper effects of mechanical stimulation versus static conditions, it provides only a partial and imperfect reconstruction of the tissue environment in vivo. At the tissue level, the detrusor and lamina propria layers of the bladder form the backbone of a tension/relaxation transfer apparatus (19). Thus, the individual cellular elements within the bladder wall exist in a complex mechanically active environment in which smooth muscle cells in particular are subjected to internal and external forces during the filling and emptying processes. The type and magnitude of the physical stimulation of bladder smooth muscle in vivo remain speculative, but the 15% cyclic strain regimen used in our in vitro studies reflects in vivo voiding pressures ranging from 80 to 120 mmH2O that the bladder experiences under pathological conditions; 80 mmHg being the threshold pressure for ischemic damage to peripheral nerves (47). These conditions might well be reflective of those in 7-day and 14-day partially obstructed rat bladders since at this stage of the obstruction, focal regions of hypoxia become apparent within the connective tissue and muscularis layers (28).
At some point during the obstruction, the bladder shifts to a decompensated state as a result of decreased blood flow and hypoxia coupled with impaired angiogenesis in the detrusor muscle (27). However, the rat model of pressure overload is more resistant than other animal models to the onset of bladder decompensation owing to a sustained blood flow to the bladder wall (27). As shown in our studies, obstruction-induced overdistension of the rat bladder activated the expression of numerous proangiogenic genes identified by microarray screening. The Cyr61, CTGF, MMP-1, MCP-1, and midkine genes were the most upregulated in mechanically challenged bladders. Surprisingly, expression of the antiangiogenic genes, IGFBP-2, IGFBP-4, and thrombospondin 1 was not affected by bladder overdistension. Similarly, the expression of type III collagen gene, which was downregulated by mechanical stretch in cultured cells, was not significantly decreased in obstructed bladders. It is clear that in vitro experiments can neither replicate the structural complexity within tissue nor are they subject to the limitations imposed by the temporal and spatial specificity of each cellular element. Indeed, obvious structural and compositional differences exist between cultured cells and tissue conditions. In addition, transcriptional regulation of gene expression in the bladder wall may be affected by diverse stimuli that coexist with mechanical overload, e.g., hypoxia, humoral factors, neurotransmitters, etc. Nevertheless, our data strongly suggest the resurgence of an active gene program of angiogenesis within the obstructed rat bladder. The reason why a complex network of mediators control angiogenesis is unknown although a high functional redundancy in this process is assumed. Similarly, the functional significance of the temporal regulation of angiogenic genes is not clear yet, but it may coincide with the vascular damages in the bladder wall and the subsequent compensatory changes within the tissue. Our histochemical analyses showed a significant decrease of capillary density in regional areas after 14 days of obstruction. Such vascular/capillary damages may be the primary cause for the activation of proangiogenic genes in obstructed tissue. Regional bladder hypoxia have been reported in obstructed bladders and drives regional angiogenesis that allowed recovery of blood supply (28). However, other studies have suggested that hypoxia limits the ability of the detrusor muscle cells to respond to, and compensate for, the alterations in their environment, further exacerbating normal functioning of the bladder (26).
Interestingly, the paradigm of mechanical overload-induced hypoxia and relationship to organ performance has been demonstrated in the cardiovascular system (46, 57). In this context, it has been demonstrated that cardiac angiogenesis, which is induced in the early adaptive phase to hemodynamic overload, is sufficient to maintain cardiac function. When myocytes enlarge without concomitant adaptive growth of capillaries, angiogenesis becomes insufficient to maintain the function of the hypertrophied heart in the maladaptive phase, presumably because of decreased expression of angiogenic factors, leading to organ failure. Micro-injection of angiogenic factors (e.g., VEGF, angiopoietin-1) directly in pressure overloaded hearts increased the number of microvessels and improved their mechanical performances. By analogy with the cardiovascular system, the use of angiogenic therapy during obstructive diseases of the bladder appears as a viable therapeutic approach for maintaining bladder wall contractility and compliance in a compensated phase and preserving its normal functioning as a result of obstructive diseases. Further studies are needed to ascertain the value of therapeutic angiogenesis in nonmalignant bladder diseases.
GRANTS
This work was supported by National Institute of Diabetes Digestive and Kidney Diseases Grants R21 DK-068483 and R56 DK-60572 (to B. Chaqour).
FOOTNOTES
Address for reprint requests and other correspondence: B. Chaqour, Dept. of Anatomy and Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Ave., Box 5, Brooklyn, NY 11203-2098 (e-mail: bchaqour{at}downstate.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
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  E. Attia, H. Brown, R. Henshaw, S. George, and J. A. Hannafin Patterns of Gene Expression in a Rabbit Partial Anterior Cruciate Ligament Transection Model: The Potential Role of Mechanical Forces Am. J. Sports Med., February 1, 2010; 38(2): 348 - 356. [Abstract] [Full Text] [PDF]
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 T. Tsuruda, K. Hatakeyama, H. Masuyama, Y. Sekita, T. Imamura, Y. Asada, and K. Kitamura Pharmacological stimulation of soluble guanylate cyclase modulates hypoxia-inducible factor-1{alpha} in rat heart Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1274 - H1280. [Abstract] [Full Text] [PDF]
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 M. Hanna, H. Liu, J. Amir, Y. Sun, S. W. Morris, M. A. Q. Siddiqui, L. F. Lau, and B. Chaqour Mechanical Regulation of the Proangiogenic Factor CCN1/CYR61 Gene Requires the Combined Activities of MRTF-A and CREB-binding Protein Histone Acetyltransferase J. Biol. Chem., August 21, 2009; 284(34): 23125 - 23136. [Abstract] [Full Text] [PDF]
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