Published online before print June 26, 2008, doi: 10.1161/CIRCRESAHA.108.179465
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© 2008 American Heart Association, Inc.
Cellular Biology |
Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine TGF-β Signaling Pathway
From the Harvard-MIT Division of Health Sciences and Technology (A.B.B., D.S.E., M.J., E.R.E.), Massachusetts Institute of Technology, Cambridge, Mass; the Cardiovascular Division (M.J.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; the Departments of Biochemistry and Ophthalmology (M.A.N.), Boston University School of Medicine, Mass; the Department of Biomedical Engineering (M.A.N.) Boston University, Mass; and the Department of Pathology, Anatomy, and Cell Biology (R.V.I.), Thomas Jefferson University, Philadelphia, Pa.
Correspondence to Aaron B. Baker, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building E25, Room 442, Cambridge, MA 02139. E-mail abbaker{at}mit.edu
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Abstract |
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Mechanical forces are potent modulators of the growth and hypertrophy of vascular cells. We examined the molecular mechanisms through which mechanical force and hypertension modulate endothelial cell regulation of vascular homeostasis. Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells. Mechanical strain stimulated the production of perlecan and heparan sulfate glycosaminoglycans by endothelial cells. By inhibiting the expression of perlecan with an antisense vector we demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control. Mechanical regulation of perlecan expression in endothelial cells was governed by a mechanotransduction pathway requiring autocrine transforming growth factor β (TGF-β) signaling and intracellular signaling through the ERK pathway. Immunohistochemical staining of the aortae of spontaneously hypertensive rats demonstrated strong correlations between endothelial TGF-β, phosphorylated signaling intermediates, and arterial thickening. Further, studies on ex vivo arteries exposed to varying levels of pressure demonstrated that ERK and TGF-β signaling were required for pressure-induced upregulation of endothelial HSPG. Our findings suggest a novel feedback control mechanism in which net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from VSMCs and growth inhibitory signals from endothelial cells.
Key Words: mechanical strain • hypertension • TGF-β • heparan sulfate proteoglycans • perlecan
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Introduction |
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The endothelium is a dynamic constituent of the vascular system that exerts remarkable control over diverse process such as hemostasis, inflammation, and the regulation of vascular tone.1,2 In addition to interacting with blood constituents and circulating cells, the vascular endothelium is exposed to a distinct mechanical environment consisting of hemodynamic shear stress and mechanical stretch from blood pressure. As a result, endothelial cells are optimally situated as both sensors and effectors in the vascular remodeling process. An extensive body of scientific work supports the concept that vascular cells respond to their mechanical environment.3,4 In vitro, shear stress and stretch stimuli regulate endothelial cell production of regulatory molecules including nitric oxide,5 reactive oxygen species,6 inflammatory cell adhesion molecules,7 and extracellular matrix molecules.8 Similarly, vascular smooth muscle cells (VSMCs) respond to mechanical stimuli by altering their expression of cell adhesion and extracellular matrix molecules9 as well as increasing proliferation and migration.10,11
An essential function of the intact endothelium is the paracrine regulation of the growth and phenotype of underlying VSMCs. Soluble heparan sulfate proteoglycans (HSPGs) are intimately involved in endothelial inhibition of VSMC proliferation.12–14 These complex molecules are composed of a core protein covalently coupled to one or more heparan sulfate glycosaminoglycan chains. The heparan sulfate chains consist of a linear polymer of alternating disaccharide units heterogenenously modified by epimerization, deacetylation, and sulfation,15 which attain an intricate molecular structure whose information capacity far exceeds that of an equivalent mass of nucleic acid polymers.16 Here, we show that HSPGs are a key component in an integrated feedback control loop regulating vascular remodeling through the modulation of paracrine endothelial inhibition of VSMC growth. Further, we define an endothelial mechanotransduction pathway that is dependent on strain-induced autocrine transforming growth factor β (TGF-β) signaling in combination with the activation of the ERK signaling pathways, and leads to alterations in expression of growth inhibitory HSPGs.
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Materials and Methods |
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Cell Culture
Human umbilical vascular endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) were purchased from Cambrex, Walkersville, Md. All VSMCs were used between passage 4 and 5 and all endothelial cultures were used at passage 3 to 5. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Stable cell lines of transfected bovine endothelial cells were as previously described.14,17
Application of Mechanical Strain to Cultured Cells
The device to apply mechanical strain to cells was as described previously.18 Briefly, 0.005'' thick silastic membranes (Specialty Manufacturing, Inc) were sterilized and coated with collagen I. Endothelial cells were grown on the collagen-coated silastic membranes and cyclic mechanical strain was applied with maximal strain of 3% or 5% at a frequency of 1 Hz. All loading experiments were performed on cells that were at least 2 days postconfluence.
Smooth Muscle Cell Proliferation Assay
Rat or human smooth muscle cells were passaged into 6-well plates at low density. Endothelial cell conditioned media was isolated and used in an assay of VSMC proliferation. Smooth muscle cells were serum starved in 0.5% calf serum for 24 hours, washed with PBS, and incubated in conditioned media with 1 µCi/mL 3H-thymidine for 24 hours. The cells were then washed 3 times with PBS at 4°C. The cells were then incubated with 10% TCA for 30 minutes at 4°C, washed twice in 95% ethanol, and solubilized in 1 mL of 0.25 mol/L NaOH with 0.1% SDS for 1 hour. The samples were then added to scintillation cocktail and radioactivity was measured using a liquid scintillation counter.
Cell Lysis and Western Blotting
Cells were lysed in 20 mmol/L Tris with 150 mmol/L NaCl and protease inhibitors. For perlecan, the samples were digested with heparitinase III and chondroitinase ABC (1 U/mL; Associates of Cape Cod) for 4 hours before Western blotting. Digested or nondigested samples were run on 4% to 15% polyacrylamide gradient gels and transferred to PVDF membranes. The membranes were blocked for 1 hour in 5% nonfat milk in PBS with 0.01% tween-20 (PBST) and exposed to the following antibodies at 4°C overnight in 1% nonfat milk: antiphospho-Smad 2 (Ser465/467), anti-Smad 2, antiphospho-p38 MAPK (Thr180/Tyr182), antip38 MAPK, antiphospho-ERK (Thr202/Tyr204), anti-ERK (Cell Signaling), mouse antiperlecan (Invitrogen), or rat antiperlecan (Laboratory Vision). The membranes were washed with PBST, incubated at room temperature for 2 hours with a secondary antibody, and detected using chemiluminescence (Perkin Elmer).
Metabolic Labeling and Isolation of Proteoglycans
At the time of mechanical loading, 100 µCi of 35SO4 and 80 µCi of 3H-glucosamine was added to each plate. After loading, the conditioned media was collected and combined with guanidine-HCl to a final concentration of 4 mol/L. The cell layers were then washed 3 times with cold PBS then PBS with 1.0 mmol/L EDTA was added and the cells were allowed to detach. The cells were spun down and washed with PBS twice and then resuspended in 1 mL of 0.05% trypsin-EDTA solution (Invitrogen) and placed on ice for 10 minutes. After the trypsin digestion, the samples were centrifuged and 1 mL of DMEM with 5% calf serum was added to neutralize the trypsin. This solution was then brought to a final concentration of 4 mol/L guanidine-HCl. Proteoglycans were extracted from the extracellular matrix for 48 hours with a solution of 50 mmol/L sodium acetate (pH=6.0), 4 mol/L guanidine-HCl, 2% triton x-100, and protease inhibitors.
ELISA Measurement of TGF-β1
The concentration of TGF-β1 was measured using ELISA (R&D Systems) according to the manufacturer’s instructions.
Animal Model of Hypertension
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Massachusetts Institute of Technology and conformed to the "Guiding Priniciples in the Care and Use of Animals" of the American Physiological Society and the NIH Guide for the Care and Use of Laboratory Animals. Age matched wild-type Wistar-Kyoto Rats and Spontaneously Hypertensive (SHR; NTac:SHR) rats were obtained from Taconic (Germantown, NY). At 20 weeks of age the animals were euthanized and the aortae were harvested.
Immunohistochemistry
The abdominal aorta from hypertensive rats was formalin fixed and sectioned using standard methods. For staining of HSPGs the slides were treated for 2 hours with a 0.4 U/mL solution of heparitinase III (Ibex, Canada). Primary antibodies were diluted in PBS containing 1% BSA, applied to slides, and incubated in a humid chamber overnight at 4°C. A monoclonal antibody recognizing "stubs" of digested heparan sulfate (3G10; Seikagaku) was used. Secondary antibody staining at detection was performed using the LSAB 2 kit (DakoCytomation) according to the manufacturer’s instructions. An AEC substrate (DakoCytomation) was used for detection of the HRP conjugate. The samples were counterstained in Mayer’s hematoxylin for 3 minutes, washed with tap water, and mounted in aqueous mounting medium (DakoCytomation). Quantitative analysis was performed using Adobe Photoshop.
Ex Vivo Perfusion System
Thoracic aortae were harvested from rats and rinsed several times in modified Krebs-Henseleit buffer with 1% penicillin-streptomycin (Sigma). The aortae were mounted in physiological orientation between 2 cannulas within the perfusion system (Figure 1). The flow system maintained the artery in a modified Krebs-Henseleit buffer with sodium biocarbonate and 5% dextran to maintain physiological viscosity. Fluid flow was driven by a peristaltic pump (Cole Palmer) and set to produce a physiological shear stress of 15 dyn/cm2 on the aortic wall. Pressure was controlled by changing the height of the fluid column following the aortic segment. High pressure was set at 200 mm Hg and normal pressure at 100 mm Hg, as measured using a pressure sensor (Harvard Apparatus). At the end of the experiment the aortic segments were frozen in isopentane cooled with liquid nitrogen.
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Statistics
Data are presented as mean±SEM. A Student t test was used to analyze for differences between 2 groups. Multiple comparisons between groups were analyzed by 1-way ANOVA followed by Student-Newman–Keuls post hoc test. A 2-tailed probability value <0.05 was considered statistical significant.
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Results |
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Cyclic Mechanical Strain Increases Endothelial Inhibition of VSMC Proliferation Through Increased Perlecan Production
One major functional role of endothelial cells is the production of soluble factors controlling VSMC growth. A fundamental question addressed by this work is how mechanical strain alters paracrine inhibition of VSMCs by endothelial cells. Endothelial cells were grown on silastic membranes for 2 days postconfluence and then exposed to uniform cyclic mechanical strain (3% or 5% maximal strain, 1 Hz) for 1 to 24 hours. Conditioned media was harvested from endothelial cells under nonstrained or strained conditions and applied to low density cultures of VSMCs (Figure 2A). In these experiments the 0% strain samples are endothelial cell conditioned media (ie, media exposed to endothelial cells without strain for the period of the experiment). Thus, the difference in DNA synthesis between control media and 0% strain samples represents the baseline inhibitory capacity of confluent endothelial cells. Brief periods of mechanical strain induced slightly stimulatory media (Figure 2B). This stimulatory effect decreased with duration of exposure to become inhibitory after 8 hours. After 24 hours of loading, mechanical strain induced a 2.3-fold increase in the inhibitory properties of the endothelial conditioned media (19.5±0.06% versus 45.6±12.7% of no strain samples; Figure 2B). Overall, conditioned media from strained endothelial cells inhibited VSMC proliferation about 80% greater than control growth media. We confirmed this effect in another endothelial cell type and found that this effect increased with the magnitude of the load (Figure 2C).
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Previous studies have suggested that perlecan can control VSMC growth.13,14 We examined the perlecan production in the conditioned media of strained and nonstrained endothelial cultures. Western blotting of the conditioned media revealed increased soluble perlecan core protein after 24 hours of loading (Figure 2D). To establish a mechanistic link between strain-induced changes in perlecan expression and inhibition of VSMCs, we applied 5% cyclic mechanical strain for 24 hours to stably transfected endothelial cell lines with either an expression vector (pcDNA3) or a vector expressing an antisense construct targeted to perlecan (Figure 2E). Transfection of this construct into endothelial cells led to a 10-fold reduction in perlecan in the conditioned media of the cells (Figure 2F). The increase in paracrine inhibition of VSMCs by endothelial cells with mechanical load was retained in endothelial cells transfected with the empty vector and eliminated in endothelial cells expressing the perlecan antisense construct (Figure 2G). To further confirm the specificity of our results we depleted endothelial conditioned media of perlecan using an antibody-based affinity column. These results also showed a reduction in the inhibition of VSMC growth after perlecan depletion (Figure 2G).
Mechanical Strain Increases Endothelial Cell Production of Soluble Heparan Sulfate Proteoglycans
The cellular production of proteoglycans can be regulated on multiple levels including direct regulation of the core protein and through alterations in the assembly the glycosaminoglycan sugar chains. We metabolically labeled the glycosaminoglycan chains during exposure to mechanical strain and found an increase of 24% in total soluble glycosaminoglycan production by HUVECs (Figure 3A and 3B). A 38% increase in soluble heparan sulfate was also observed. With strain, no change in cell surface total glycosaminoglycan was observed, but a decrease in cell-associated heparan sulfate was found. Total glycosaminoglycans and heparan sulfate glycosaminoglycans in the extracellular matrix were increased 85% and 20%, respectively, for mechanically strained versus nonstrained cultures.
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Mechanical Strain Controls Perlecan Expression Through ERK and p38 MAPK-Dependent Autocrine TGF-β Production
TGF-β is intimately involved in regulating cell growth and production of extracellular matrix.19 We examined the amount of TGF-β produced by endothelial cells under mechanical strain in the presence of inhibitors to the ERK and p38 MAPK signaling pathways. In endothelial cells, TGF-β production was increased by exposure to mechanical strain (Figure 4A). This effect was significantly blocked by both inhibitors to MEK1/2 (U0126) and p38 MAPK (SB029063). We also hypothesized that because mechanical strain induced TGF-β production, an autocrine signaling pathway through this growth factor may be an important control mechanism. To examine the intracellular signaling pathways responsible for mechanical strain–mediated modulation of HSPGs, we pretreated cells with inhibitors to the ERK and p38 MAPK signaling pathways. After inhibitor or neutralization antibody treatment we used Western blotting to measure the levels of phosphorylated signaling intermediates, including Smad 2 (a downstream effector of TGF-β signaling), p38 MAPK, and ERK1/2 (Figure 4B). Mechanical strain led to activation of these intermediates after 24 hours. Interestingly, there was crosstalk between the signaling pathways. Maximal Smad 2 phosphorylation only occurred in the absence of both inhibitors to the ERK and p38 MAPK signaling cascades. Further, phosphorylation of ERK1/2 and p38 MAPK was partially blocked by inhibitors to each of the other pathways. We performed a similar Western blotting analysis in mechanically stimulated cultures pretreated with a neutralizing antibody to TGF-β. These studies demonstrated that TGF-β was required for optimal phosphorylation of the intracellular signaling pathways (Figure 4C).
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We also examined the role of these pathways in the production of the heparan sulfate proteoglycan core proteins in response to mechanical strain. Inhibitors to p38 MAPK, MEK1/2, and the neutralizing antibody to TGF-β attenuated the load-induced increase in perlecan production (Figure 4D and 4E). To confirm the involvement of the TGF-β signaling pathway, we added TGF-β added before mechanical strain and found that TGF-β increased perlecan expression independent of mechanical load (Figure 4F). Further, we blocked TGF-β signaling using a TGF-β receptor I inhibitor and found no increase in perlecan expression in the presence of this inhibitor with mechanical strain (Figure 4F). Measurements of cellular cytotoxicity were made under the various treatment conditions using an LDH release assay indicating no cellular toxicity under long-term treatment with these inhibitors and mechanical strain (data not shown).
Spontaneously Hypertensive Rats Have Increased Endothelial HSPGs, TGF-β, and ERK Signaling
To further extend our work to in vivo models of altered hemodynamics, we examined the association between vascular remodeling, heparan sulfate, and intracellular signaling pathway intermediates in an animal model of hypertension (Figure 5). Histological staining and morphological analysis revealed increased medial thickening in spontaneously hypertensive rats (SHR) in comparison to wild-type (WKY) rats. In SHR and WKY animals the tunica media and endothelial layer stained heavily for HSPGs. The specificity of staining for HSPGs was confirmed by lack of staining in arterial sections not digested with heparitinase but stained with primary and secondary antibodies (data not shown). The endothelial cell HSPG staining increased by 58% in SHR rats compared to WKY controls. We also found increases in endothelial TGF-β, phospho-Smad 2, phospho-ERK, and phospho-p38. Interestingly, TGF-β and phospho-Smad 2 were reduced in the medial layers of the aorta. We then examined the association between arterial remodeling (media thickening) and the various signaling pathways (Table). This analysis revealed strong correlations between medial thickness, p38, ERK, and TGF-β in the endothelium.
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Pressure-Induced Changes in Endothelial HSPGs Require Autocrine TGF-β and ERK Signaling
To further verify that these pathways were acting in an intact arterial system we created a culture system allowing the application of pulsatile pressure and flow to ex vivo rat aortae. As the maintenance of endothelial integrity was an important factor in our experiments, we confirmed endothelial integrity by immunostaining for PECAM in the arteries after 24 hours of culture (Figure 6A). In this system we demonstrated that 24 hours of high pulsatile pressure (mean pressure of 200 mm Hg) induced increased expression of endothelial HSPG, TGF-β, phospho-Smad 2, and phospho-ERK in comparison to arteries exposed to low pulatile pressure (mean pressure of 100 mm Hg; Figure 6B and 6C). Inhibitors to TGF-β and MEK1/2 (U0126) blocked high pressure–induced expression of endothelial HSPG. Interestingly, the inhibitors of the ERK pathway also inhibited TGF-β production and phospho-Smad 2 upregulation. Similarly, the neutralizing antibody to TGF-β blocked the phosphorylation of ERK. In contrast to our in vitro studies on HUVECs and in hypertensive animals, the p38 pathway did not appear to be activated in ex vivo arteries by 24 hours of load (data not shown). In addition, inhibitors to this pathway did not inhibit load induced HSPG expression or activation of the TGF-β and ERK pathways. Together these results are consistent with and support a model of autocrine TGF-β signaling linked to ERK signaling as the control mechanism for pressure induced modulation of endothelial HSPGs.
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Discussion |
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Mechanical forces have long been known to be potent regulators of vascular endothelial function.3 Endothelial cells have evolved sophisticated sensory and regulatory ability to maintain vascular homeostasis through adaptive remodeling.20 This study addresses the question of how endothelial cells respond to mechanical strain to control the growth of the underlying VSMCs. Previously, it was known that endothelial cells can regulate VSMC proliferation.21 In particular, heparin and endothelial cell HSPGs are potent inhibitors of VSMC proliferation and FGF-2 induced mitogenesis.13,22–24 This regulation is growth state dependent, with subconfluent cultures of endothelial cells stimulating VSMC growth and postconfluent cultures inhibiting VSMC growth.12,25–28 Similarly, perlecan and endothelial-derived HSPGs have been shown to be essential in inhibiting the neointimal response to vascular injury.14,29–31 Our study adds a new dimension to these results, demonstrating that the regulation of perlecan by mechanical strain is an important mechanism in altering endothelial paracrine inhibition of VSMC proliferation in response to changes in mechanical environment. Further, we have also demonstrated a mechanotransduction pathway in endothelial cells controlling their production of perlecan in response to mechanical forces.
Our in vitro studies with cultured endothelial cells indicate that optimal stimulation of perlecan production requires both ERK and p38 MAPK signaling as well as autocrine TGF-β signaling. In turn, p38 MAPK signaling is dependent on ERK and autocrine TGF-β. TGF-β production and subsequent stimulation of phospho-Smad 2 is partially blocked by inhibitors of both of these pathways. We therefore propose an expanded model of endothelial cell control of vascular homeostasis in which ERK and p38 signaling are important for activation of TGF-β, and TGF-β drives the continued stimulation of ERK and p38 pathways. Consistent with this model, inhibitors of these intracellular signaling pathways or TGF-β signaling significantly reduced mechanical strain-stimulated increases in perlecan production. This paradigm is also supported by the correlations found between TGF-β and ERK/p38 in the endothelium of the SHR rats. Further correlations between members of a TGF-β autocrine signaling loop and vascular wall thickness are consistent with a shared mechanistic pathway.
Ex vivo studies demonstrated that pressure-induced upregulation in HSPG expression in the endothelium was also blocked by inhibitors to both ERK and TGF-β. Interestingly, in our ex vivo system the p38 pathway did not appear to be regulated by high pressure and was not required for load-induced increases in HSPG expression. Several factors could account for the relative insensitivity of the p38 pathway to pressure and its nonessential role in mechanical regulation of HSPG expression. Acute strain ex vivo and wall strain in chronic hypertensive rat models may differentially affect p38, the drugs used to inhibit p38 may be less stable in the ex vivo setting or may act on VSMCs as well as endothelial cells in the excised artery. In vitro, in vivo, and ex vivo studies were consistent in supporting the central nature of TGF-β autocrine signaling in combination with ERK signaling to control HSPG production and ultimately modulation of VSMC proliferation.
In the context of the intact arterial system, the finding that endothelial cells produce more inhibitory factors to VSMCs with mechanical load is in contrast with the behavior of VSMCs under similar mechanical strain conditions. Mechanical load induces VSMC proliferation,10,11 release of FGF-2,32 production of PDGF,33 and elaboration of cell surface associated HSPG.34,35 Consequently, we propose that under mechanical stimulation the endothelium provides negative feedback control inhibiting the progrowth signals produced by VSMCs under mechanical strain. In the absence of an intact endothelium, as might arise after vascular injury or disease, mechanical forces would induce VSMCs to produce growth stimulatory factors that are unchecked by load-enhanced endothelial cell paracrine growth inhibition. Once the endothelium is restored, additional means of responding to hemodynamic forces can be applied including regulation of vascular tone and inhibition of VSMC proliferation. In this context, our findings contribute to the understanding of the complex coregulation of these 2 cell types under hemodynamic stimuli and during aberrant vascular remodeling in the face of persistent injury imposed by chronic hypertension and indwelling endovascular devices.
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Acknowledgments |
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We gratefully acknowledge the technical assistance of P. Seifert and G. Wong. Additionally, we thank Dr Martha Gray for the loan of the mechanical loading device.
Sources of Funding
This work was supported by U.S. National Institutes of Health (NIH) grant R01 HL49039 (to E.R.E) and a Postdoctoral Research Fellowship from Philip Morris U.S.A. (to A.B.B.).
Disclosures
None.
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Footnotes |
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Original received December 11, 2007; resubmission received May 14, 2008; revised resubmission received June 13, 2008; accepted June 17, 2008.
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References |
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