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Am J Physiol Cell Physiol 292: C488-C496, 2007. First published August 30, 2006; doi:10.1152/ajpcell.00337.2005
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VASCULAR BIOLOGY

Biomechanical regulation of hedgehog signaling in vascular smooth muscle cells in vitro and in vivo

David Morrow,1 Catherine Sweeney,1 Yvonne A. Birney,1 Shaunta Guha,1 Nora Collins, Philip M. Cummins,1 Ronan Murphy,1 Dermot Walls,2 Eileen M. Redmond,3 and Paul A. Cahill1

1Vascular Health Research Centre, Faculty of Science and Health, and 2School of Biotechnology and the National Centre for Sensor Research, Dublin City University, Dublin, Ireland; and 3Department of Surgery, University of Rochester Medical Center, Rochester, New York

Submitted 8 July 2005 ; accepted in final form 23 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hedgehog (Hh) signaling has recently been shown to be both responsive to mechanical loading in vitro and to control vascular development in vivo. We investigated the role of cyclic strain and pulsatile flow in modulating Hh signaling and growth of adult rat vascular smooth muscle cells (SMC) in culture. Exposure of SMC to defined equibiaxial cyclic strain (0% and 10% stretch, 60 cycles/min, for 24 h) significantly decreased sonic hedgehog (Shh) and patched 1 (Ptc1) expression while concurrently inhibiting Gli2-dependent promoter activity and mRNA expression, respectively. Cyclic strain significantly decreased SMC proliferation (cell counts and proliferating cell nuclear antigen expression) concomitant with a marked increase in SMC apoptosis (fluorescence-activated cell sorter analysis, acridine orange staining of apoptotic nuclei and Bax/Bcl-xL ratio). These strain-induced changes in proliferation and apoptosis were significantly attenuated following addition of either recombinant Shh (3.5 µg/ml) or overexpression of the Notch 3 intracellular domain (Notch IC). Further studies using a perfused transcapillary culture system demonstrated a significant decrease in Hh signaling in SMC following exposure of cells to increased pulsatile flow concomitant with a decrease in proliferation and an increase in apoptosis. Finally, the pulsatile flow-induced decreases in Hh signaling were validated in vivo following flow-induced rat carotid arterial remodeling after 28 days. These data suggest that Hh expression is diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling and atherogenesis in vivo.

cyclic strain; apoptosis; proliferation

HEMODYNAMIC FORCES associated with the flow of blood play an important role in the physiological control of vascular tone, remodeling, and associated vascular pathologies. These forces include pulsatile flow-induced cyclic circumferential strain, which is caused by a transmural force acting perpendicular to the vessel wall (10, 19, 23, 36). Transduction of biomechanical stimuli leads to activation of cellular signaling mechanisms that ultimately lead to adaptive, and sometimes maladaptive, changes in cell and tissue fate (21, 29). The ultimate arbiter of vascular cell fate (growth, migration, differentiation, and apoptosis) in response to hemodynamic stimulation is unclear but considered fundamental to the pathogenesis of vascular disease. Strain-induced changes in smooth muscle cell (SMC) growth, defined as the balance between SMC proliferation and apoptosis, participates in the local vascular reaction to hypertension, late lumen loss and restenosis after vascular interventions, as well as plaque vulnerability during atherosclerosis (10, 19, 23, 36). Since changes in vascular cell fate are also apparent during vascular morphogenesis and modeling of the embryonic vasculature (21, 29), the control of these cell fate decisions in adult cells may share similar signaling patterns.

Hedgehog (Hh) genes are a class of 19-kDa morphogens that interact with heparin on the cell surface through an NH2-terminal basic domain and are tethered to the surface through cholesterol and fatty acyl modification (4, 13). There are three human homologues of the Drosophila Hh gene: sonic Hh (Shh), desert Hh, and Indian Hh. Of these, Shh is the most widely expressed during development, and lack of Shh is embryonically lethal with multiple defects in early to mid-gestation (5, 30). Hh ligands can signal in at least two ways; they may be tethered to the plasma membrane of the signaling cell to effect short range signaling, or they may be released from the signaling cell in a diffusible form to act as a long-range signal, where release is regulated by the Dispatched protein (4, 13). The Hh protein, either released or tethered, serves as a ligand for receptors located on the membrane of responding cells (4, 13). Signaling by all three Hh proteins occurs through interaction with the patched receptors (Ptc1 and 2) that then activate the transcription factors Gli1, Gli2, and Gli3. The downstream targets of the Gli gene products include both Ptc and Gli (4, 13). In the absence of an Hh ligand, Ptc1 functions catalytically to inhibit an effector molecule called smoothened (Smo) and prevent downstream signaling. In the presence of ligand, Ptc1 transduces the Hh signal by releasing its inhibition of Smo (4, 13). Several recent observations highlight the involvement of Hh in the development of embryonic vascular tissues, including hypervascularization of neuroectoderm following overexpression of Shh (34), disorganization of endothelial precursors in Shh-deficient zebrafish (5), and poor vascularization of the developing lung in Shh-deficient mice (30). A study by Pola et al. (32) showed that Shh signaling is present in adult cardiovascular tissues and can be activated in vivo to induce robust angiogenesis.

Hh upregulates the expression of Notch target genes during arterial differentiation suggesting a role for Shh in maintaining cell proliferation through the Notch signaling pathway (43). Notch signaling regulates intercellular communication and directs individual vascular cell fate decisions (14, 20). The intracellular portion of Notch, Notch IC, is translocated to the nucleus, where it interacts with the CSL family of transcription factors [CBF-1/RBP-Jk, Su(h), and LAG-1] to become a transcriptional activator that can then modulate the expression of Notch target genes "Hairy Enhancer of Split" (hes) gene and HES-related transcription factors (Hrts) (11, 15). Recent studies demonstrate that Notch receptors and hrt genes are coordinately upregulated in neointimal cells but downregulated in medial cells following vascular injury, an effect that is mimicked by the addition of serum mitogens (PDGF) to cultured cells (40, 42) and by cyclic strain in vitro (27). Furthermore, Notch signaling may be a critical determinant of SMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis (39, 41, 42).

Biomechanical signals induce a highly restricted transcriptional response in vascular SMC that include genes that can modify vascular structure (10, 19, 23, 36). Hh is a critical mediator in transducing mechanical signals to stimulate chondrocyte proliferation in vitro (47). We therefore examined the specific role of biomechanical stimulation of adult SMC in controlling endogenous Hh signaling components and their contributory role in controlling the growth response of these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All items were of the highest purity commercially available and purchased from Sigma Aldrich (Poole, Dorset, UK) unless otherwise stated.

Cell culture. Rat vascular SMC (R354-05) were purchased from Cell Applications and grown in culture as previously described (27, 39). Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg streptomycin in a humidified atmosphere of 5% CO2-95% air at 37°C. Cells were routinely subcultured using 0.125% trypsin-EDTA at 37°C. SMC displayed typical spindle shaped morphology and hill and valley pattern of growth in culture and stained positively for smooth muscle cell specific {alpha}-actin.

Cyclic strain studies. For cyclic strain studies, cells were seeded into 6-well Bioflex plates (Dunn Labortechnik, Asbach, Germany) at a density of ~6 x 105 cells/well. When cells had reached ~80% confluency, a Tension Plus FX-4000T system (Flexcell International, Hillsborough, NC) was subsequently employed to apply a physiological level of cyclic strain to each plate (0–10% strain, 60 cycles/min, 0–24 h with the use of the Heartbeat simulation).

Perfused transcapillary co-culture system. The perfused transcapillary culture apparatus (Cellmax Quad artificial capillary culture system, Spectrum Laboratories; see Refs. 8, 33) consisted of an enclosed bundle of 50 semipermeable Pronectin-coated polypropylene capillaries (13 cm capillary length, 330 µM internal diameter, 150 µM thick wall, 0.5 µm pore size, 100 cm2 extracapillary surface area, and 70 cm2 lumenal surface area) through which medium from a reservoir is pumped, at a chosen flow rate, via silicone rubber tubing. As the gear pump rotates, the motor shaft forces the pump pins to depress the pump tubing on the capillary module, thereby forcing culture media to flow in a pulsatile fashion through the gas-permeable silicone flow path tubing into the capillary. By altering the flow rate using an electronic control unit that is housed outside the humidified incubator, varying pulsatile flow rates and hence pulse heights (pressure) can be achieved in this system. To maintain pH, PCO2, and PO2 of the culture media at constant levels, the perfused transcapillary culture system was housed in a humidified atmosphere in a standard CO2 incubator, thereby allowing gaseous exchange to occur through the silicone rubber tubing. Cells (1 x 108 cells) were seeded and grown on the outside of each capillary for 7–10 days and perfused at a rate of 0.3 ml/min before pulsatile flow rates were ramped. Pulse pressures were monitored simultaneously intraluminally at the inlet port and extraluminally at the side port using pressure transducers connected to a recorder (models 7 and 7E, Grass Instruments). In the current study, the "low" pulsatile flow rate used was 0.3 ml/min, corresponding to a pulse pressure of 24/18 mmHg, a frequency of 0.2 Hz, and an amplitude of 6 mmHg in the extracapillary space. The "high" pulsatile flow rate was 25 ml/min, corresponding to a pulse pressure of 64/14 mmHg, a frequency of 2 Hz, and an amplitude of 50 mmHg in the extracapillary space. Cellular mRNA was harvested using TRIzol (Invitrogen) according to the manufacturer's specifications.

Biomechanical induced vascular remodeling in vivo. Flow-induced vascular remodeling was performed through blood flow restriction in the left common carotid, as previously described (26). Briefly, Sprague-Dawley rats were anesthetized before their internal and external carotid arteries were ligated. The common left carotid artery (LCA) blood flow was reduced to that of the patent occipital artery. The LCA and right carotid artery (RCA) blood flows were measured before the ligation and again 3 and 28 days post ligation with the use of an ultrasonic transit-time volume flow meter (Transonic Systems). Sham-operated animals were used as controls. Vessels were harvested and examined for Hh components.

Notch and hedgehog expressing vectors/luciferase reporter plasmids. Shh expression vector consists of full-length mouse Shh cDNA (250 bp 5' UTR, 1,314 bp coding, and 1 Kb 3' UTR) clone and was a kind gift of Pao-Tien Chuang of the Cardiovascular Research Institute, University of California, San Francisco. The Gli2-Lc luciferase reporter plasmid (pGL3b/8xGli-lcLuc) was a kind gift of Philip A. Beachy, Johns Hopkins University School of Medicine (Baltimore, MD). Notch 3 IC expression vector (CMX-Notch 3 IC) and CBF-1/RBP-Jk inhibitor, RPMS-1 were all kind gifts, as previously reported (27, 39).

Plasmid preparation, transient transfection, luciferase, and beta-galactosidase assays. Plasmids were prepared for transfection according to the manufacturer's instructions using a Qiagen plasmid midi kit (Qiagen, Crawley, UK), as described previously (19, 21). Cells were plated onto 6-well plates 2 days before transfection, at a density of 1 x 105 cells/well, and transfected at 70% confluency. Plasmid transfection was performed with the use of Lipofectamine Reagent (Invitrogen). The cells were transfected with a luciferase reporter construct, and various expression constructs, with the addition of a total of 2.0 µg DNA/well. Transfection efficiency was confirmed and normalized to beta-galactosidase activity following co-transfection with pCMV-LacZ (a plasmid-encoding beta-galactosidase activity). Western blot analysis was also performed, whenever possible, to confirm over expression of effector proteins. Cells were harvested 16–24 h post transfection, using 1x Reporter Lysis Buffer (Promega, Madison, WI). Transactivation of reporter genes was evaluated by the luciferase assay (Promega) and normalized to the beta-galactosidase activity. The latter was performed according to the manufacturer's instructions (high-sensitivity beta-galactosidase assay; Stratagene, La Jolla, CA). To maximize the number of cells encoding each plasmid-encoded vector, transfected cells were puromycin selected and pooled as previously described (27, 39).

Western blot analysis. Proteins from cell lysates (12–15 µg) were resolved on SDS-PAGE (12% resolving, 5% stacking) before transfer onto nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). Membranes were stained in Ponceau S to ensure equal protein loading and rinsed in wash buffer (PBS containing 0.05% Tween 20) before being probed as described previously (27, 39).

Immunocytochemistry. SMC were seeded onto 6-well plates 2 days before being stained at 2 x 105 cells per well. Cells were stained for Shh at 80–90% confluency using the following protocol. Cells were washed 3 times in 1x PBS. The cells were then permeabilized and fixed in methanol (–20°C, 10 min), and subsequently rehydrated in 1x PBS/3% BSA (10 min). Cells were then incubated in the appropriate primary antibody (1:50 dilution in 1x PBS/3% BSA) at 4°C overnight with gentle agitation. Following three 10-min washes in 1x PBS, cells were incubated in the appropriate secondary antibody (1:200 dilution in 1x PBS/3% BSA using FITC or anti-goat AlexaFluor) for 2–3 h at 37°C. Cells were then washed once in 1x PBS before visualization with the use of an Olympus DP-50 fluorescent microscope, using appropriate excitation and emission spectra at x20 magnifications.

Acridine orange/ethidium bromide cell staining. Apoptotic cells were stained and visualized using an acridine orange/ethidium bromide stain, as described previously (27, 39).

Apoptosis assay by fluorescence-activated cell sorter analysis. Apoptotic effects of treatments were determined using the Vybrant Apoptosis Assay Kit (Molecular Probes, Bio Sciences), followed by fluorescence-activated cell sorter (FACS) analysis using a FACScan flow cytometer (Becton Dickinson, Dublin, Ireland). Cells were designated as viable, apoptotic, or necrotic.

Quantitative real-time RT-PCR. Quantitative real-time RT-PCR was carried out using the Rotor Gene (model RG-3000; Corbett Research, Sydney, Australia) and the SYBR green PCR kit (Qiagen), as described previously (21). The gene-specific oligonucleotide sequences were the following: Ptc1, forward, 5'-GCT GGA GGA GAA CAA GCA AC-3' and reverse, 5'-CCA GGA GTT TGT AAG CGA GG-3'; Smo, forward, 5'-AAT TGG CCT GGT GCT TAT TG-3' and reverse, 5'-CTG AAG GTG ATG AGC ACG AA-3'; Gli2, forward, 5'-CGC CTG GAG AAC TTG AAG AC-3' and reverse, 5'-TTC TCA TTG GAG TGA GTG CG-3'; Shh, forward, 5'-CCT TTA GCC TAC AAG CAG TT-3' and reverse, 5'-GGC ATT TAA CTT GTC TTT GC-3'.

Shh activation/inhibition. For hedgehog signaling activation, cells were treated for 24 h with 3.5 µg/ml of recombinant Shh protein (R&D Systems, Abingdon, UK) or transfected with a Shh expression vector encoding full-length mouse Shh cDNA. For inhibition studies, cells were treated with 40 µM cyclopamine reagent (Biomol Research Laboratories, Plymouth Meeting, PA). Control cells were also treated with vehicle control (dimethyl formamide).

Data analysis. Results are expressed as means ± SE. Experimental points were performed in triplicate, with a minimum of three independent experiments. Unpaired Student's t-test and Wilcoxon's signed-rank test were used for comparison of the two groups. A value of P ≤ 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Shh stimulates SMC growth and inhibits SMC apoptosis in vitro. Immunocytochemical analysis, Western blot analysis, and real time-quantitative real time (QRT)-PCR of steady-state mRNA levels in static cells confirmed the presence of components of the Hh signaling pathway in adult rat vascular SMC in culture. Static SMC expressed both protein and mRNA for Shh, its receptor, Ptc1, Smo, and the downstream target gene Gli2 (Fig. 1A). Immunocytochemical staining for Shh was observed within the cytoplasm while a discrete vesicular pattern of intracellular lysosomal localization of native Ptc1 was evident in these cells (Fig. 1A). Activation of Hh signaling with plasmid-encoded Shh significantly increased Smo and Gli2 mRNA levels after 24 h, an effect that was significantly attenuated following inhibition of Shh signaling with cyclopamine (Fig. 1B). In parallel cultures, recombinant Shh (3.5 µg/ml) stimulated SMC clonal proliferation after 5 days in culture while plasmid-encoded Shh increased cell number after 9 days, an effect that was attenuated following inhibition of Shh signaling with cyclopamine (Fig. 1C). In contrast, recombinant Shh (3.5 µg/ml) decreased the number of apoptotic nuclei in serum-deprived SMC while inhibition of endogenous Shh signaling with cyclopamine significantly increased the number of apoptotic nuclei in serum-stimulated cells (Fig. 1D).


Figure 1
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  		Fig. 1. Hedgehog (Hh) components in rat vascular smooth muscle cells (SMC) in vitro. A: representative immunocytochemical analysis and immunoblot of sonic hedgehog (Shh) and patched receptor 1 (Ptc1) expression in vascular SMC. The immunoglobin AlexaFluor control showed no discernible staining. Representative agarose gel electrophoresis of RT-PCR amplification of Shh, smoothened molecule (Smo), and Gli2 mRNA transcripts in SMC. B: effect of Shh overexpression with plasmid-encoded Shh on Smo and Gli2 mRNA levels in SMC using quantitative real time (QRT)-PCR in the absence or presence of Hh inhibitor cyclopamine (40 µM). Data are representative of three independent experiments with similar results. C: effect of recombinant Shh (3.5 µg/ml) on clonal cell proliferation and plasmid encoded Shh on serum-stimulated SMC growth in the absence or presence of cyclopamine (40 µM). The cumulative data represent the mean values from 3 independent experiments ± SE; *P ≤ 0.05 vs. mock control. D: effect of Hh activation with recombinant Shh (3.5 µg/ml) or Hh inhibition with cyclopamine (40 µM) on SMC apoptosis. Representative data of acridine orange/ethidium bromide stained positive cells (apoptotic cells). Cumulative data on the number of apoptotic nuclei in serum-deprived and serum-stimulated cells after 48 h in the absence or presence of recombinant Shh (3.5 µg/ml) or cyclopamine (40 µM), respectively. FCS, fetal calf serum. The cumulative data represents the mean values from 3 independent experiments ± SE. *P ≤ 0.05 vs. control.

 
Cyclic strain modulates SMC growth (proliferation and apoptosis) while decreasing Shh signaling in vitro. The lack of an effect of cyclic strain on SMC viability was confirmed by measuring LDH levels in media from strained cells (data not shown). Cyclic strain (10%, 1 Hz for 24 h) significantly increased the number of apoptotic nuclei (Fig. 2A) concomitant with an increase in the Bax/Bcl-xL ratio by increasing the mRNA levels of the pro-apoptotic protein, Bax, and decreasing the mRNA levels of the anti-apoptotic protein Bcl-xL (Fig. 2B) compared with unstrained cells. In addition, cyclic strain (10%, 1 Hz for 24 h) significantly decreased the number of SMC after 10 days in culture (Fig. 2C), compared with unstrained cells.


Figure 2
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  		Fig. 2. Cyclic strain-induced changes in SMC fate and Hh signaling. A: cumulative data on the percentage of apoptotic nuclei in unstrained (0%) and strained (10%) cells for 24 h. B: cumulative data on the %change in Bax and Bcl-xL mRNA expression ratio in serum-stimulated and serum-deprived cells following exposure to 10% equibiaxial strain for 24 h. C: data were normalized to GAPDH and represent the mean values from 3 independent experiments ± SE; *P ≤ 0.05 vs. unstrained control cyclic strain-induced decrease in SMC growth. Serum-stimulated SMC growth following exposure to 0% (unstrained) and 10% equibiaxial strain for 24 h before cell counts were determined on day 6 post strain. Cyclic strain-induced changes in Shh and Ptc1 protein expression (D), Shh, Smo, Ptc1, and Gli2 mRNA (E), and Gli2 (F) promoter transactivation after exposure to 0% (unstrained) and 10% equibiaxial strain for 24 h. Data were corrected for total protein using a ponceau stain for protein and GAPDH for mRNA, respectively. Representative immunoblot and cumulative data from three independent experiments ± SE. *P ≤ 0.05 vs. unstrained control. Luciferase assays were normalized to beta-galactosidase activity.

 
In parallel cultures, cyclic strain (10%, 1 Hz, 24 h) caused a significant decrease in Shh protein expression concomitant with a significant decrease in the expression of Ptc1, a Hh target gene (Fig. 2D). Cyclic strain also significantly decreased Shh, Smo, Ptc1, and Gli2 steady-state mRNA levels compared with unstrained controls (Fig. 2E). Further analysis of Hh target gene expression using the Gli2-luciferase-tagged promoter revealed that cyclic strain caused a significant decrease in serum-stimulated Gli2 transactivation compared with unstrained controls (Fig. 2F).

Effect of recombinant Shh on cyclic strain-induced changes in Hedgehog signaling in vitro. The cyclic strain-induced changes in SMC growth and apoptosis were examined following treatment of the cells with recombinant Shh for 24 h. In the presence of recombinant Shh (3.5 µg/ml), Gli2 mRNA levels were significantly enhanced in strained and unstrained cells (Fig. 3A). However, while the direction of the strain-induced response was unaltered by Shh treatment, the extent of strain-induced inhibition of Gli2 mRNA levels was such that following Shh treatment, the Gli2 mRNA levels returned to levels comparable to that of unstrained cells (Fig. 3A).


Figure 3
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  		Fig. 3. The effect of recombinant Shh on strain-induced changes in SMC proliferation and apoptosis. The effect of recombinant Shh (3.5 µg/ml) on cyclic strain-induced changes in (A) Gli2 mRNA (B) pCNA levels after 24 h. Data were corrected for total protein using a ponceau stain or GAPDH using QRT-PCR. Representative immunoblot and cumulative data on the percent inhibition of Gli2 mRNA and pCNA expression in the absence or presence of Shh (3.5 µg/ml). Cumulative data are from 3 independent experiments ± SE. *P ≤ 0.05 vs. unstrained control; #P ≤ 0.05 vs. strain (–) Shh. The effect of recombinant Shh (3.5 µg/ml) on cyclic strain-induced apoptosis after 24 h. C: representative data showing discrete populations of cells distinguished using flow cytometry designated as viable (V), early apoptotic (EA), late apoptotic (LA), or necrotic (N), depending on the AnnexinV/propidium iodide (PI) staining. D: cumulative data are from 3 independent experiments ± SE. *P ≤ 0.05 vs. unstrained control; $P ≤ 0.05 vs. strain (–) Shh.

 
Cell proliferation was monitored by measuring proliferation cell nuclear antigen (pCNA) levels in both unstrained and strained cells following Shh treatment. Recombinant Shh (3 µg/ml) increased pCNA levels in both unstrained and strained cells (Fig. 3B). However, the strain-induced decrease in pCNA levels in the absence of Shh was significantly greater than the decrease in expression following treatment of cells with recombinant Shh (Fig. 3B).

The cyclic strain-induced changes in SMC apoptosis were also examined following treatment of the cells with recombinant Shh for 24 h. Apoptosis was monitored using an annexin V/propidium iodide FACS-based apoptotic assay in both unstrained and strained cells following Shh treatment (3.5 µg/ml). Control experiments were performed with SMC following serum treatment (10% FCS) and serum deprivation (0.2% FCS) for 24 h to demonstrate the sensitivity of the assay (Fig. 3C). Recombinant Shh (3.5 µg/ml) decreased the number of apoptotic nuclei in both unstrained and strained cells (Fig. 3C). However, the strain-induced increase in apoptosis in the absence of Shh was significantly greater than the increase in the number of apoptotic nuclei following treatment of cells with recombinant Shh (Fig. 3D).

Effect of overexpression of the intracellular domain of the Notch 3 receptor on cyclic strain-induced changes in Hedgehog signaling in vitro. We have previously shown that cyclic strain regulates SMC fate, in part, through regulation of Notch receptor and downstream target gene expression (27). Moreover, Notch 3 IC receptor overexpression significantly recovered the strain-induced changes in SMC proliferation and apoptosis to levels comparable with unstrained control cells (27). We therefore examined the effects of constitutively active Notch 3 IC on Hh signaling in these cells. Over expression of constitutively active Notch 3 IC was confirmed using an anti-hemagglutinin antibody specific for cells expressing the hemagglutinin-tagged plasmid encoding Notch 3 IC (data not shown). Enforced expression of Notch 3 IC significantly increased Gli2 promoter transactivation and steady state mRNA levels compared with mock controls (Fig. 4A). Furthermore, inhibition of Notch dependent CBF-1/RBP-Jk signaling following coexpression with RPMS-1 resulted in a marked decrease in Notch 3 IC-induced Gli2 promoter activity and mRNA levels in these cells (Fig. 4A).


Figure 4
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  		Fig. 4. The effect of constitutive overexpression of Notch 3 IC on strain-induced changes in Hh signaling. The effect of over expression of constitutively active Notch 3 IC on Gli2 promoter transactivation (A) and Gli2 mRNA levels (B) following inhibition of CBF-1/RBP-Jk dependent signaling with coexpression of RPMS-1. The effect of over expression of constitutively active Notch 3 IC on Hh target gene expression in unstrained and strained cells. QRTPCR analysis of Gli2 (C) and Ptc1 (D) mRNA levels following overexpression of constitutively active Notch 3 IC in unstrained (0% strain) and strained (10% strain). Data were normalized to GAPDH. The cumulative data represents the mean values from three independent experiments ± SE, **P ≤ 0.05 vs. unstrained mock control; *P ≤ 0.05 vs. strained mock control; $P ≤ 0.05 vs. unstrained Notch 3 IC control.

 
In parallel studies, over expression of constitutively active Notch 3 IC recovered the strain-induced decreases in Hh signaling (Fig. 4B), while concomitantly recovering the strain-induced changes in SMC growth and apoptosis to levels comparable to unstrained controls (27). Specifically, the strain-induced decrease in Gli2 and Ptc1 mRNA levels in mock-transfected cells were significantly greater than the strain-induced decrease following overexpression of Notch IC (Fig. 3B).

Pulsatile flow inhibits SMC growth while attenuating hedgehog signaling in vitro and in vivo. With the use of a perfused transcapillary culture system, chronic exposure of SMC to pulsatile flow (low: 0.3 ml/min, 6 mmHg, 0.5 dyn/cm2 and high: 25 ml/min, 56 mmHg, 23 dyn/cm2) for 72 h resulted in a significant decrease in SMC proliferation with a concurrent increase in SMC apoptosis. This was evident by a significant decrease in cell numbers and a concurrent increase in the expression of apoptotic markers, cellular apoptosis susceptibility, p53 and Bax, respectively (Fig. 5A). In parallel cultures, exposure of SMC to pulsatile flow also resulted in a significant decrease in Hh target gene steady-state mRNA levels, namely, Smo, Gli2, and Ptc1 (Fig. 5B).


Figure 5
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  		Fig. 5. Pulastile flow-induced Hh signaling in vitro and in vivo. A: pulsatile flow-induced changes in SMC proliferation and apoptosis. Serum-stimulated SMC proliferation and apoptosis (CAS, p53, and Bax expression) following exposure to low and high pulsatile flow rates (low: 0.3 ml/min; 6 mmHg; 0.5 dyn/cm2 and high: 25 ml/min; 56 mmHg; 23 dyn/cm2) for 72 h. Representative blot and cumulative data are the means ± SE of 3 independent experiments; *P ≤ 0.05 vs. low flow. B: pulsatile flow-induced changes in Hh signaling. QRT-PCR analysis of SMC Smo, Ptc1, and Gli2 mRNA following exposure to low and high flow for 72 h. The cumulative data represents the mean values from 3 independent experiments ± SE; *P ≤ 0.05 vs. low flow control. C: pulsatile flow-induced changes in Hh signaling in vivo. Data represent the carotid blow flow measurements and Shh and Ptc1 expression following ligation of the left carotid artery after 28 days. Data were corrected for total protein using a Ponceau stain. The cumulative data represents the mean values from 5 vessels ± SE. PCNA, proliferating cell nuclear antigen; LCA, left carotid artery; RCA, right carotid artery.

 
To determine whether similar changes in Hh signaling occur during flow-induced arterial remodeling in vivo (26), we examined the effects of flow on Hh signaling in the rat carotid ligated artery. The acute and chronic effects of ligation of the LCA on changes in blood flow were measured. Blood flow was significantly decreased by >90% in LCA, while a compensatory increase in blood flow of 80 ± 5% occurred in the contralateral RCA (Fig. 5C). Examination of the extent of Hh signaling in both the LCA and RCA after 28 days revealed that Shh and Ptc1 expression was significantly reduced under high flow conditions compared with low flow (Fig. 5C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study examined for the first time the effect of biomechanical stimulation on Hh signaling in adult SMC and determined the contributory role of Hh in regulating changes in SMC proliferation and apoptosis. We established that cyclic strain induces a significant decrease in the pro-proliferative and anti-apoptotic effects of Hh by decreasing the expression and activity of components of the Hh signaling pathway. The readdition of recombinant Shh recovered the expression of Hh target gene expression to levels comparable to that of unstrained cells while concurrently attenuating the strain-induced changes in SMC proliferation and apoptosis. Since enforced regulation of the intracellular domain of the Notch 3 receptor recovers SMC cell growth and apoptosis in strained cells (27), we further examined whether Notch signaling could recover Hh signaling and hence fate in these cells. Overexpression of constitutively active Notch 3 IC reversed the strain-induced inhibition of Hh signaling to levels comparable to that of unstrained cells while concurrently attenuating the strain-induced SMC growth and apoptosis in vitro (19). Similarly, exposure of SMC to changes in pulsatile flow resulted in a marked diminution of Hh signaling concomitant with a decrease in cell growth (8) and an increase in SMC apoptosis (2). Moreover, the importance of these findings was confirmed in vivo using a model of carotid flow-induced arterial injury, where Hh signaling was diminished significantly in the contralateral (high flow) arteries. Collectively, these studies suggest for the first time that biomechanical stimulation of SMC inhibits endogenous Hh signaling resulting in fundamental changes in vascular SMC proliferation and apoptosis in vitro and in vivo. Moreover, these data further reinforce the importance of the Hh/Notch axis in controlling vascular cell fate decisions in vitro in response to strain/pulsatile flow-induced pressure. Understanding these responses may provide new insights into the pathogenesis and treatment of vascular diseases, such as atherosclerosis and intimal hyperplasia.

Since Hh signaling plays a key role in pattern formation, differentiation, and proliferation in the early mouse embryo, the vascular system was overlooked as a target of Hh action when knockout mutants of individual Hh genes were initially examined (7). Nevertheless, a role for Hh signaling in blood vessel formation in the embryo is now supported by several observations, including the hypervascularization of neurectoderm in response to overexpression of Shh (34) and the decreased vascularization of lung tissue in Shh-deficient mice (30). More extensive studies (7) have focused on differentiation of the axial vessels in the zebrafish embryo, yolk sac angiogenesis and endothelial tube formation in the mouse embryo, and angiogenesis in the adult mouse. To the best of our knowledge, our study is the first to demonstrate functional Hh signaling in vascular SMC in vitro. Activation of Hh signaling with recombinant Shh and/or plasmid encoded Shh resulted in a significant pro-proliferative anti-apoptotic response while inhibition of endogenous Hh signaling with cyclopamine resulted in a reciprocal response in vitro. In addition, Shh and the Hh target gene, Ptc1 were both expressed in carotid arteries and were differentially regulated following vascular remodeling in vivo. It is also now clear in vivo that the adult vascular system can respond functionally to Hh (31, 32). Ptc1-LacZ expression, an indicator of Hh response in Ptc1-LacZ heterozygous mice, is measurable in both endothelial cells and adventitial fibroblasts that surround the vessels, with the fibroblasts capable of robust Ptc1 upregulation in response to administered Hh (32). In a hindlimb ischemia model, Shh treatment promotes an increase in capillary density and blood flow (32). Ptc1 is upregulated in the interstitial mesenchymal cells and a Shh-blocking antibody inhibits angiogenesis (31). Shh addition also promotes neovascularization and the formation of large, well-branched vessels in a corneal angiogenesis assay (31).

Hemodynamic forces associated with the flow of blood play an important role in the physiological control of vascular remodeling and associated vascular pathologies (1, 19, 23, 36). The present study examined the specific role of endogenous Hh signaling components in controlling mechanosensitive regulation of SMC fate in vitro. To this end, we used two in vitro models of mechanotransduction (1, 8) and validated these data using an in vivo model of flow-induced vascular injury (6, 26). While previous studies have presented conflicting reports on the effects of cyclic strain on SMC growth in vitro (3, 12, 17, 35, 37, 38, 45), it is clear that SMC can either increase or decrease their proliferative capacity. In the current study, cyclic strain decreased SMC proliferation while concomitantly increasing SMC apoptosis as described previously by our laboratory (27) and others (24, 35). This concurs with several studies that report increased SMC apoptosis in vivo (25, 28, 44) in response to strain or pressure. The cyclic strain-induced SMC apoptosis is, at least in part, associated with a change in the Bax/Bcl-xL ratio in favor of apoptosis. Similarly, exposure of SMC to changes in pulsatile flow, and hence pulse pressure, resulted in a similar antiproliferative (8) proapoptotic response in these cells, with a change in the Bax/Bcl-xL ratio also in favor of apoptosis (2).

Our data supports a functional role for biomechanical-induced decreases in Hh-dependent signaling and subsequent changes in vascular cell fate. Cyclic strain increased SMC apoptosis and decreased SMC proliferation concomitant with a marked decrease in Hh signaling in these cells. In a similar manner to strain, pulsatile flow decreased Hh signaling concomitant with a decrease in cell growth and an increase in apoptosis (2, 8). The level of inhibition of Hh signaling following exposure to cyclic strain and pulsatile flow suggests that Hh is a major target for biomechanical regulation of SMC in vitro. Moreover, recombinant Shh recovered the expression of Hh target gene expression to levels comparable to that of unstrained cells while concurrently attenuating the strain-induced changes in SMC proliferation and apoptosis. As cyclic strain also decreases Notch signaling to govern cell fate (27), overexpression of Notch 3 IC in strained cells recovers the growth and apoptotic response of these cells to levels comparable to unstrained cells (27) while concurrently recovering Hh downstream signaling (Gli2 and Ptc1) to levels comparable to unstrained cells. These data further reinforce the importance of the Hh/Notch axis in controlling vascular cell fate decisions in vitro. In addition, our in vivo data further validate the importance of biomechanical-induced Hh signaling, since Ptc1 expression, an indicator of the Hh response, significantly decreases in high-flow environments in vivo when changes in SMC cell fate are apparent (26). These data also suggest that the changes in Hh signaling are dependent on changes in flow since the flow rates are significantly different between the LCA and contralateral RCA.

While the location of these changes was not specifically addressed, it is likely that the predominant changes in Hh signaling occur within the medial layer in vivo. However, a role for the adventitial layer cannot be ruled out since Ptc1-LacZ expression in Ptc1-LacZ heterozygous mice is specifically measurable in adventitial fibroblasts that surround the vessels, with the fibroblasts capable of robust Ptc1 upregulation in response to administered Hh (32). Nonetheless, to our knowledge, this is the first demonstration that the expression of Hh and downstream target genes are directly modulated by strain and pulsatile flow in mammalian cells in vitro and in vivo. This finding further suggests a possible nexus by which the activation of a biomechanical signaling pathway is coupled to a cellular fate program through engagement of Hh. Additional studies will be required to delineate the precise mechanism(s) by which cyclic strain and pressure regulate the expression of Hh.

There has been to date no data supportive of a role of mechanosensitive Hh signaling in vascular cells. However, it is clear that Hh is a critical mediator transducing mechanical signals to stimulate chondrocyte proliferation (46, 47). Several recent studies confirm that Indian Hh may transduce mechanical signals during cartilage growth and repair processes (46, 47). This induction is abolished by gadolinium, an inhibitor of stretch-activated channels (18). In the present study, exposure to cyclic strain for 24 h resulted in a decrease in Gli2 promoter transactivation concomitant with a reduction in Hh target gene expression. While an acute induction of Shh signaling and downstream target gene expression (Ptc1 or Gli2) was not evident in our in vitro studies, it is notable that biomechanical activation of SMC in vivo resulted in a dramatic decrease in Hh components after 28 days. Whether an initial increase in Hh target gene expression is present, it is clear that diminution of Hh signaling is a critical mediator transducing mechanical signals to control SMC growth after chronic exposure to changes in cyclic strain and pulsatile flow when significant changes in SMC cell fate are apparent (26).

The regulation of Hh signaling is known to occur at multiple levels including patterns of ligand and receptor expression, Hh-ligand interactions and covalent modifications including phosphorylation (4, 13). One possible mechanistic explanation for the effect of cyclic strain and/or pressure on Hh signaling may be posttranslational modifications such as phosphorylation that may influence its transactivation capacity (16, 18). Indeed, glycogen synthase kinase-3 (GSK3) is known to modulate Hh signaling through phosphorylation of Gli (9). In the absence of the Hh signal, the kinases protein kinase A, GSK3, and casein kinase 1 phosphorylate Gli and mediate its degradation to the repressor form. Moreover, the regulatory phosphorylation of GSK3 and hence its activity is under the control of MAPK-dependent signaling pathways (22). It is therefore tempting to speculate that cyclic strain modulates this process through mechanosensitive regulation of MAPK thereby influencing the repressor function of Gli. Moreover, it is possible that cyclic strain may inhibit endogenous inhibitor(s) of Hh, which result in a loss of Hh functionality post strain. These possibilities among others are currently under investigation.

In conclusion, we have shown for the first time, in vitro and in vivo, that biomechanical stimuli induce significant downregulation of Hh signaling concomitant with significant changes in SMC growth and apoptosis. Biomechanical-induced changes in SMC fate are attributable, at least in part, to biomechanical inhibition of Hh signaling and can be recovered following Shh treatment or constitutive overexpression of Notch 3 IC, which can compensate for the force induced changes in Hh signaling and SMC fate (27). These studies provide initial evidence for biomechanical regulation of Hh signaling pathways in vascular cells in vitro and further suggest a critical role for Hh in determining vascular cell fate in vivo.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by grants from the Higher Education Authority of Ireland (PRTLI Cycle III), Wellcome Trust, Science Foundation Ireland, the Health Research Board of Ireland (to P. A. Cahill), and by National Institutes of Health Grants HL-59696 and AA-12610 (to E. M. Redmond).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Cahill, Faculty of Science and Health, Vascular Health Research Centre, Glasnevin, Dublin 9, Ireland (e-mail: paul.cahill{at}dcu.ie)

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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 DISCUSSION
 GRANTS
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