Pericyte Rho GTPase Mediates Both Pericyte Contractile Phenotype and Capillary Endothelial Growth State

【摘要】  Pericytes regulate microvascular development and maturation through the control of endothelial cell motility, proliferation, and differentiation. The Rho GTPases have recently been described as key regulators of pericyte shape and contractile phenotype by signaling through the actin cytoskeleton in an isoactin-specific manner. In this report, we reveal that Rho GTPase-dependent signal transduction not only influences pericyte shape and contractile potential but also modulates capillary endothelial proliferative status and pericyte-endothelial interactions in vitro. We provide evidence that overexpression of mutant Rho GTPases, but not other Ras-related small GTPases, significantly alters pericyte shape, contractility, and endothelial growth state in microvascular cell co-cultures. In particular, we describe the use of a silicon substrate deformation assay to demonstrate that pericyte contractility is Rho GTP- and Rho kinase-dependent; further, we describe a novel in vitro system for examining pericyte-mediated endothelial growth arrest and show that control pericytes are capable of growth-arresting capillary endothelial cells in a cell contact-dependent manner, whereas pericytes overexpressing dominant-active and -negative Rho GTPase are comparably incompetent. These data strongly suggest that signaling through the pericyte Rho GTPase pathway may provide critical cues to the processes of microvascular stabilization, maturation, and contractility during development and disease.
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Development, maturation, and remodeling of the vascular system is a multistage process with regulatory mechanisms at each step.1 Several perivascular cell types play major roles in the modulation of microvascular maturation and contractility, including the smooth muscle cells associated with arteries and the pericytes associated with venules and capillaries.2,3 Perivascular cell regulation of the capillary microenvironment occurs through dynamic maintenance of the basement membrane as well as regulation of microvascular tone, through a complex array of signaling intermediates.4 A complete understanding of vascular development, the physiology of capillary tone, and the regulation of capillary permeability provides insight into the pathophysiology of the vascular dysfunction associated with tumor angiogenesis,5 age-related macular degeneration,6 and diabetic retinopathy,7 as well as the physiological angiogenesis of wound healing.8
The microvascular pericyte in particular has been the subject of considerable experimental interest because of its role in regulation of microvascular endothelial growth and differentiation9 as well as capillary contractility and microvascular tone.10 In particular, through both pericyte-endothelial cell contact-dependent as well as endothelial-independent mechanisms, pericytes have been postulated to govern the phenotypic change from a proliferative angiogenic sprout to a mature microvascular conduit with a quiescent capillary endothelium.11,12 Both direct evidence for pericyte suppression of endothelial growth13 and migration14 as well as in situ correlation between pericyte investment and vessel stability have been reported.11,15 Interestingly, pericyte investment has been implicated in conferring capillary stability and resistance to regression in vivo,16,17 suggesting that pathological angiogenesis requires previously quiescent endothelium to destabilize its association with pericytes and re-enter the cell cycle. Although recent work in tumor biology has also highlighted pericytes as a novel drug target that may enhance the efficacy of anti-angiogenic chemotherapy,18-21 the precise molecular and biochemical mediators regulating pericyte-dependent microvascular remodeling remain equivocal.
Evidence is accumulating that vascular morphogenesis may be regulated by members of the Rho family of small GTPases.22 Upstream of multiple cytoskeletal and kinase-mediated effectors, Rho GTPases control physiological maintenance of arterial tone, as well as the dysregulation and hypertrophic remodeling associated with essential hypertension.23,24 Importantly, a recent role for Rho GTPase-dependent signal transduction has now been suggested in the control of pericyte shape and contractility, leading to microvascular tone and blood flow regulation via alterations in cytoskeletal dynamics.25
In this study, we demonstrate that pericyte-specific and Rho GTPase-dependent signal transduction reversibly regulates both pericyte contractility and capillary endothelial cell growth state. Building on the previous work of Harris et al26 and others, we have developed two novel in vitro systems to directly quantify and simultaneously link the contractile potential of microvascular pericytes with pericyte Rho GTPase-mediated endothelial cell growth control. In these systems, we alter pericyte Rho GTPase expression via both adenoviral-mediated gene delivery and direct transfection of dominant-active or -negative Rho constructs. Results reveal that increased signaling through the Rho GTPase pathway significantly augments pericyte contractility and impairs pericyte efficacy in inducing endothelial cell growth arrest through both contact-dependent and contact-independent pericyte-endothelial interactions. Therefore, alterations in Rho GTPase-dependent signal transduction specifically modulate pericyte shape and contractile phenotype, as well as regulate their ability to control endothelial growth. This lends support for the notion that pathological angiogenesis is linked to alterations in endothelial growth state downstream of signaling aberrations within microvascular pericytes.

【关键词】  pericyte mediates pericyte contractile phenotype capillary endothelial

Materials and Methods

Cell Culture

Bovine retinal pericytes (expressing vascular smooth muscle actin, NG2 proteoglycan, and 3G5) and endothelial cells (expressing CD31, von Willebrand factor, and demonstrating uptake of acetylated low-density lipoprotein) were isolated from neonatal cow retina as previously described27 and used through passage three on tissue culture-treated plasticware (Corning, Inc., Corning, NY) in Dulbecco??s modified Eagle??s medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% bovine calf serum (Hyclone, Logan, UT), supplemented with penicillin, streptomycin, and Fungizone (Invitrogen). Cells were grown in 24-well tissue culture plates (Corning, Inc.) in a total volume of 1 ml unless otherwise noted.

Recombinant Adenoviruses and Infection

Adenoviruses expressing dominant-active and dominant-negative Rho GTPase under the control of a tetracycline transactivator were obtained from Daniel Kalman (Emory University School of Medicine, Atlanta, GA). The viruses were amplified in human embryonic kidney 293 cells and purified by freeze/thaw and centrifugation. Expression of each virus was tested by infection of COS7 cells for 12 hours at multiplicities of infection of 100 to 500 followed by immunoblot of cell lysates and immunofluorescence microscopy with anti-Rho antibodies (clone 26C4; Santa Cruz Biotechnology, Santa Cruz, CA; data not shown). In the experiments detailed here, pericytes were infected with dominant-active or dominant-negative Rho GTPase-containing viruses in combination with the transactivator virus in serum-containing media for 6 hours at optical density-determined multiplicities of infection of 216, 298, and 286 for dominant-active Rho, dominant-negative Rho, and tetracycline transactivator-containing virus, respectively.

Plasmids and Transfection

Dominant-active Ras in vector pZipNeo (pZipNeoRasL61) was the generous gift of Dr. Deniz Toksoz (Tufts University School of Medicine, Boston, MA). Dominant-active Rac1 (pMT3RacL61) and dominant-active Cdc42 (pMT3Cdc42L61) in vector pMT3 were contributed by Dr. Larry Feig (Tufts University School of Medicine, Boston, MA). Green fluorescent protein (GFP)-expressing plasmid (pEGFP-N3) was purchased from Clontech (Palo Alto, CA). Pericytes were transfected with 0.8 µg of DNA per coverslip for 24 hours per the Effectene transfection reagent protocol (n > 6 for each condition; Qiagen, Valencia, CA).

Rho GTPase Small Molecule Inhibitor

The pyridine derivative (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632; Sigma-Aldrich, St. Louis, MO), previously shown to specifically inhibit the activity of p160 Rho-associated kinase at an IC50 of 140 nmol/L,28 was resuspended in sterile water, stored at 10 mmol/L, and used at final concentrations ranging from 1 to 10 µmol/L.

Pericyte-Endothelial Cell Co-Culture Assay

Co-Culture

Pericytes were plated onto UV-sterilized 12-mm circular glass coverslips (Fisher, Pittsburgh, PA) and placed in 24-well tissue culture plates (Corning, Inc.) at a density of 10,000 cells/well in complete 10% bovine calf serum containing DMEM for 24 hours, followed by infection with recombinant adenovirus at multiplicities of infection of 200 to 300 as above in complete 10% bovine calf serum containing DMEM for 6 hours. After infection, cells were washed and incubated for an additional 18 hours before the addition of 10,000 cells/well of freshly trypsinized bovine retinal endothelial cells in complete 10% bovine calf serum containing DMEM. After 20 hours of co-culture, media was supplemented with 10 µg/ml 5'-bromodeoxyuridine (BrdU; Sigma-Aldrich). At 24 hours of total co-culture, cells were prepared for immunocytochemistry.

Immunocytochemistry

Coverslip-attached co-cultures were fixed in 4% paraformaldehyde containing serum-free DMEM for 5 minutes at room temperature. Cells were permeabilized for 90 seconds at room temperature in freshly prepared 50 mmol/L HEPES/50 mmol/L PIPES buffer supplemented with 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 75 mmol/L KCl, and 0.1% Triton X-100. Chromosomally incorporated BrdU epitopes were unmasked by incubation for 15 minutes at 37??C in 1 N HCl in phosphate-buffered saline (PBS). Primary antibody incubation was performed at 4??C in a humidified chamber overnight; fluorophore-tagged secondary antibody incubation was performed at room temperature in a humidified chamber for 45 minutes. Nuclei were counterstained with 1:1000 Hoechst no. 33258 (Sigma-Aldrich) in PBS.

Antibodies

Staining was performed using anti-smooth muscle actin (SMA) (clone 1A4; Biogenex Laboratories, San Ramon, CA), anti-BrdU (BD Pharmingen, San Diego, CA), and anti-Myc (clone A14; Santa Cruz Biotechnology) primary antibodies and Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies (Invitrogen) diluted 1:200 in PBS/azide. Visualization was performed using an Axiovert 200M microscope (Carl Zeiss, Thornwood, NY) at x400, and image analysis was performed with Metamorph software (Universal Imaging Corp., Downingtown, PA).

Quantification

Pericytes were identified by immunoreactivity for SMA; dominant-active Rho GTPase-infected pericytes were identified by additional immunoreactivity for a Myc epitope tag, whereas dominant-negative Rho GTPase-infected pericytes were identified based on previously described morphology.25 In particular, Rho-inhibited pericytes possess a highly spread-out morphology with enlarged surface area, as well a characteristic radial pattern of phalloidin-staining with markedly diffuse central vascular smooth muscle actin staining. Contact between pericytes and endothelial cells was confirmed at x630 magnification with high-resolution optics (NA = 1.4). Co-cultures were assessed for endothelial proliferative index by examining pericyte-contacting versus lone SMA-negative endothelial cells and scoring each subset for proliferating versus nonproliferating cells assessed by nuclear BrdU immunoreactivity. Observers scored experiments by alphanumeric code, blinded to experimental condition. The number of proliferating individual endothelial cells in contact with processes from a single pericyte was scored and compared with lone endothelial cells with no cell-cell contacts; endothelial cells in contact with other endothelial cells or multiple pericytes as well as endothelial cells with pyknotic or atypical nuclei were excluded from analysis. Endothelial proliferative index was calculated as the percentage of BrdU-positive endothelial cells versus total endothelial cells in each subset. Experiments were performed at least three times in triplicate, with >300 cells assessed per condition; results are expressed as mean percentages ?? SE.

Analysis of Pericyte Contractile Phenotype

Preparation of Deformable Silicone Substrates

Deformable silicone substrates were prepared essentially as previously described.26 In brief, 20 to 50 µl of dimethylpolysiloxane (no. 123K0769; Sigma-Aldrich) were pipetted onto 35-mm round glass coverslips using a positive displacement pipettor. The silicone substrate was permitted to spread at room temperature before heat cross-linking by passing the silicone-coated coverslip through a Bunsen burner flame. Silicone-coated coverslips were then placed within a glow discharge apparatus,29 generating a plasma discharge onto the silicone to create a hydrophilic surface permitting protein adsorption. Coated coverslips were then incubated with 0.1% collagen type I in PBS to facilitate subsequent cell attachment.

Quantitation of Contractile Force Production in Pericytes

Pericytes expressing virally transduced mutant Rho GTPases or empty vector control plasmids as well as cells treated with pharmacological inhibitors were plated on deformable silicone substrates after viral infection or as a function of drug treatment as described above. Silicone-attached pericytes were grown in cell culture chambers enabling live cell viewing for protracted time periods. The rate and extent of silicone substrate deformation was then quantified using standard morphometric analyses applied to phase-contrast images taken using a Zeiss Axiovert 200M computer-assisted light microscope imaging workstation. Each experiment was performed at least three times in triplicate; mean percentages of cells wrinkling ?? SE were graphed as a function of Rho GTPase and Rho kinase inhibitor treatments.

Results

Alteration of Rho GTPase Signaling in Pericyte Cell Culture

In vivo, pericytes regulate microvascular dynamics through multiple different mechanical and biochemical pathways; however, these pathways are as yet poorly elucidated. To more closely examine the role that the Rho GTPase signaling cascade plays in pericyte function and microvascular physiology, we used both adenovirus-based overexpression of dominant-active or dominant-negative Rho GTPase mutants in conjunction with a specific small molecule inhibitor to alter signaling through Rho GTPase and its downstream effector, Rho kinase. Not only were the downstream effects of mutant Rho GTPase overexpression characterized, but Rho GTPase-overexpressing and Rho kinase-inhibited pericytes were assayed for pericyte contractile phenotype and endothelial proliferation. Results of these experiments reveal that perturbation of the Rho GTPase signaling pathways significantly influences pericyte contractile phenotype, as well as endothelial cell growth state.

Previous work indicated that transient transfection of mutant Rho GTPases alters pericyte cytoskeletal array and cell shape by signaling through the actin network in an isoform-specific manner.25 To expand on this, we used a drug-selectable, adenoviral-mediated gene delivery system to specifically induce mutant Rho GTPase expression in microvascular pericytes. Dominant-active (Myc-tagged) and dominant-negative (untagged) Rho GTPase expression was controlled using an adenoviral delivery system under the control of a tetracycline-repressible promoter (Tet).30,31 Pericytes were plated and allowed to adhere for 24 hours before infection with altered Rho GTPase constructs; after a second 24-hour period of growth, cells were fixed and stained for -SMA. As shown in Figure 1 , pericytes expressing the constitutively active (GTP-bound) form of Rho GTPase (RhoDA) as verified by immunoreactivity for the Myc epitope tag seem hypercontractile, containing numerous actin-enriched projections surrounding a centrally contracted cytoplasmic mass . This is consistent with previously published observations using transient transfection of altered Rho GTPase constructs, reporting smaller average cell size as well as formation of pronounced stress fibers.25 In contrast, pericytes transduced with a dominant-negative Rho GTPase (irreversibly locked in an inactive GDP-bound form, RhoDN) appear as flattened and polygonally shaped cells (Figure 1B) , with a -actin-containing cytoskeleton unaffected by the GDP-bound Rho GTPase overexpression; this is consistent with previous published characterization of the dominant-negative Rho phenotype as possessing significantly larger average cell size and a cytoskeletal architecture rich in phalloidin-staining stress fibers.25 This morphology is easily distinguished from the reduced surface area, condensed shape, and diffuse attenuated expression of -SMA observed in the dominant-active Rho GTPase-transduced cell cultures (Figure 1, A and B) . Tetracycline transactivator-infected (Tet) and control uninfected pericytes retain the characteristic wild-type morphology, with isoactin cytoskeletons harboring significant -SMA stress fiber expression (Figure 1B) .

Figure 1. Adenoviral alteration of Rho GTPase signaling modulates pericyte morphology. A: Bovine retinal pericytes were incubated for 24 hours, co-infected with adenoviruses containing dominant-active Rho GTPase under the control of a tetracycline-repressible transactivator for 6 hours, washed with fresh media, and incubated for an additional 18 hours. Cultures were then fixed and stained for the presence of the Myc epitope tag and for -smooth muscle actin with parallel phase images as labeled. B: Bovine retinal pericytes were infected as above with adenoviruses containing control tetracycline-repressible transactivator (Tet) alone, dominant-active Rho GTPase and Tet (RhoDA), or dominant-negative Rho GTPase and Tet (RhoDN), or mock-infected (control). Cultures were then fixed and stained for the presence of -smooth muscle actin (green), with Hoechst nuclear counterstaining (blue), with parallel phase images. Arrows indicate a stably infected RhoDA pericyte; arrowheads indicate a stably infected RhoDN pericyte. Original magnifications: x200 (A); x100 (B).

Rho GTPase Signaling Control of Pericyte Contractile Phenotype

Given the morphological differences between dominant-active and -negative Rho GTPase-expressing pericytes, we hypothesized that these differences represent a Rho-dependent change in contractile potential as well. We quantified contractile force production by using a cell culture-treated deformable silicon substrate as described in detail in the Materials and Methods section. Dominant-active (RhoDA), dominant-negative (RhoDN), or control-infected pericytes (Tet) as well as noninfected wild-type pericytes (control) plated on these silicone substrata were mounted into a temperature-controlled cell culture chamber permitting light microscopic visualization during culture. Force transduction was monitored dynamically by quantifying the percentage of cells able to generate sufficient force to produce substrate deformation visible by light microscopy (Figure 2 , arrowheads indicate wrinkle-producing contractile cells). Consistent with the above-described morphological data, dominant-active Rho-infected pericytes demonstrated a contractile, substrate-deforming phenotype with 1.5-fold greater frequency compared with vector-infected and uninfected pericytes (RhoDA 74.12 ?? 3.13%, P < 0.05 compared with either Tet or control). Conversely, dominant-negative Rho-infected pericytes generated sufficient contractile force to produce a substrate-deforming phenotype at 25% of the control frequency (RhoDN 12.4 ?? 1.81%, P < 0.05 compared with either Tet or control). Vector alone-infected pericytes were similar to uninfected controls, with baseline contractile frequencies of 52.66 ?? 3.51% and 48.98 ?? 3.48%, respectively.

Figure 2. Adenoviral alteration of Rho GTPase signaling modulates pericyte contractility. A: Bovine retinal pericytes were transduced with adenoviruses containing dominant-active (RhoDA) or dominant-negative (RhoDN) Rho GTPase mutants, tetracycline-repressible transactivator (Tet) alone, or mock-infected (control). After incubation for 24 hours after infection, cells were trypsinized and replated onto plasma glow discharge-prepared, type I collagen-coated silicon substrates. Cultures were monitored in a modified cell culture container allowing real-time phase-contrast imaging during culture. Representative phase-contrast images are provided as labeled, where arrows indicate substrate-wrinkling, actively contractile pericytes. Original magnifications, x400. B: At 24 hours, contractility was assessed by the number of pericytes producing visible substrate wrinkling per each condition, expressed as mean percentages ?? SE (n > 100 cells per condition, triplicate experiments.

To localize more finely the downstream effectors responsible for contractile phenotype regulation in Rho GTPase-altered pericytes, we treated stably infected cells with the small molecule Rho kinase inhibitor Y-2763228,32 and reassessed their contractile state (Figure 3) . Consistent with Rho GTPase control of contractile phenotype in wild-type pericytes, substrate-wrinkling frequency was reduced from baseline to complete extinction throughout the micromolar range of Y-27632 concentration in a dose-dependent manner (Figure 3 , arrows indicate wild-type wrinkle-producing contractile cells). Whereas 8 µmol/L Y-27632 is required to completely abolish contractile activity in pericytes overexpressing dominant-active Rho, 50% less Y-27632 (4 µmol/L) is sufficient to extinguish contractility in dominant-negative Rho-expressing pericytes. Indeed, treatment of dominant-active Rho-expressing pericytes with Y-27632 completely abrogates the hypercontractile state in a dose-dependent manner. The Y-27632 dose required to inhibit the steady state contractility of wild-type and vector-transduced pericytes is equivalent (Figure 3) .

Figure 3. Wild-type and Rho GTPase-mutant pericyte contractility is dose dependently reversible by small molecule inhibition of Rho kinase. A: Bovine retinal pericytes were transduced with adenoviruses containing dominant-active Rho GTPase (RhoDA), dominant-negative Rho GTPase (RhoDN), transactivator alone (Tet), or mock-infected (control) as above. After 24 hours of culture, cells were trypsinized and replated onto plasma glow discharge-prepared, type I collagen-coated silicon substrates in media containing 0 to 10 µmol/L of the small molecule Rho kinase inhibitor Y-27632, and monitored by real-time phase-contrast imaging. Representative phase-contrast images of inhibitor-treated wild-type, mock-infected pericytes are provided as labeled, where arrows indicate substrate-wrinkling, actively contractile pericytes. Original magnifications, x400. B: At 24 hours, contractility was assessed by the number of pericytes producing visible substrate wrinkling per each condition, expressed as mean percentages ?? SE (n > 100 cells per condition, triplicate experiments.

Rho GTPase Signaling Control of Pericyte-Mediated Endothelial Cell Growth Arrest

In addition to revealing the role that Rho GTPase signaling plays in controlling pericyte shape and contractile phenotype, we further investigated whether perturbations in Rho GTPase-dependent signal transduction are similarly instrumental in endothelial growth control. To determine whether perturbation of the Rho GTPase signaling pathway altered the pericyte??s ability to modulate capillary endothelial growth state, we developed a co-culture assay in which contact-dependent and -independent pericyte-mediated endothelial growth control could be quantitatively assessed. In this assay, Rho-altered pericytes are co-cultured with capillary endothelial cells; cell-cell contact and proliferative status are quantified, allowing assessment of the effects of both pericyte contact and pericyte-derived soluble mediators on endothelial proliferation (Figure 4A) .

Figure 4. Adenoviral alteration of pericyte Rho (but not Ras, Rac1, or Cdc42) GTPase signaling impedes pericyte-mediated endothelial cell growth arrest. Bovine retinal pericytes were transduced with adenoviruses containing dominant-active Rho GTPase (RhoDA), dominant-negative Rho GTPase (RhoDN), transactivator alone (Tet), or mock-infected (control) as above and co-cultured with bovine retinal endothelial cells for 24 hours. BrdU was incorporated into the co-culture medium for the last 4 hours. A: Dominant-active Rho-infected pericyte co-cultures were then fixed and stained for the pericyte marker cytoplasmic -smooth muscle actin and proliferative endothelial cell nuclear BrdU incorporation (SMA + BrdU, red), the Myc epitope tag as a marker of infection for RhoDA (Myc, green), and nuclei by Hoechst (Hoechst, blue). Parallel phase images are provided. Arrows: A BrdU-positive, pericyte-contacting proliferating endothelial cell. Arrowheads: A lone BrdU-negative, quiescent endothelial cell. B: Adenoviral Rho GTPase-altered pericytes as above, as well as dominant-active Ras, Rac1, Cdc42, and GTP control-transfected pericytes, were co-cultured for 24 hours with bovine retinal endothelial cells as above. Co-cultures were then scored for nuclear BrdU-positive endothelial proliferation as a function of pericyte contact and GTPase status. Results are expressed as mean percentages ?? SE (n > 400 cells/condition, experiments in triplicate). *P < 0.05 for differences between lone and pericyte-contacting endothelial cells with same GTPase status; P < 0.05 for differences between lone endothelial cell populations with different Rho GTPase status. Original magnifications, x200.

In parallel with control of pericyte morphology and contractility, alterations in Rho GTPase signaling also caused significant abrogation of pericyte-mediated endothelial growth arrest (Figure 4B) . At baseline, control and uninfected pericytes demonstrated significant inhibition of proliferation in pericyte-contacting versus lone endothelial cells by 49.9 ?? 13.5% and 44.0 ?? 9.7%, respectively (n = 636 and 719, P < 0.05 for both conditions). In comparison, both dominant-active and -negative Rho-expressing pericytes failed significantly to contact-inhibit adjacent endothelial cells, with contacted endothelial cell proliferation rates 1.22 ?? 0.08 and 1.28 ?? 0.09 times that of lone endothelial cells, respectively (n = 917 and 888). Interestingly, the endothelial cell proliferation rate was significantly higher in nonpericyte-contacting endothelial cells cultured with Rho-altered pericytes (50.7 ?? 3.1% for RhoDA, 53.9 ?? 6.0% for RhoDN) compared with vector and uninfected controls (26.6 ?? 2.7% and 30.0 ?? 3.6%, respectively; P < 0.05 compared with either Rho-altered pericyte condition). Both of these effects were unique to Rho among small GTPases, as epitope tag-confirmed transfection of pericytes with dominant-active forms of the Ras, Rac, and Cdc42 small GTPases (see Materials and Methods) showed intact pericyte contact-mediated growth arrest by an average of 55.8 ?? 2.2% (Ras 53.9 ?? 5.0%, Rac 55.3 ?? 4.4%, and Cdc42 58.2 ?? 4.1%; n = 479, 533, and 478, respectively), with no significant alteration in pericyte-mediated endothelial growth arrest compared with wild-type pericytes (60.0 ?? 10.9%, n = 515). This parallels previous results indicating that Rho plays a unique role among the small GTPases in the modulation of isoactin array.25

Discussion

Here, we demonstrate that Rho GTPase-dependent signaling plays a pivotal role in multiple facets of pericyte biology; in particular, using both adenoviral infection and plasmid transfection of dominant-active and -negative Rho constructs, we directly demonstrate that Rho GTPase pathway signaling through the isoactin network significantly alters Rho kinase-dependent cellular contractility. Moreover, using a novel in vitro assay of endothelial cell growth arrest, we demonstrate that pericyte regulation of microvascular endothelial proliferative status is impaired by either augmenting or diminishing Rho GTPase signaling. These effects are not reproduced by inhibition of signaling through other Ras-related small GTPases, including Ras, Rac, and Cdc42, expanding on previous work demonstrating that GTP-bound Rho, but not other Ras-related GTPases, specifically regulates pericyte contractility in an isoactin-specific manner.25

Mechanochemical Signaling in Pericytes

Our studies reveal key relationships between GTP-dependent signaling pathways and the microvascular pericyte cytoskeleton and its contractile effectors. The transforming growth factor (TGF)-ß/SMAD signaling pathway has been implicated in retinal pericyte morphology and function,33,34 as well as in pericyte-mediated retinal endothelial cell survival signaling.35 Together, these data extend earlier observations implicating TGF-ß as a key mediator of pericyte control of the endothelium.36-38 Surprisingly, our data reveal that the proliferative index of nonpericyte-contacted endothelial cells in co-culture with both dominant-active and -negative Rho-infected pericytes is significantly increased above that of controls. This indicates the possibility that altered pericyte Rho expression may also alter secretion of a soluble mediator controlling endothelial proliferation. Therefore, we assayed by enzyme-linked immunosorbent assay levels of both total and activated TGF-ß present in pericyte-conditioned culture media, as well as assessed for the presence of dominant-negative-acting soluble TGF-ß receptor II into the media by Western blotting. Neither differences in levels of active or latent TGF-ß nor the presence of soluble TGF-ß receptor II was detected in dominant-active and -negative Rho-infected pericyte conditioned media (M.E.K., J.T. Durham, and I.M.H., unpublished observations). These findings are consistent with a recent study by Kondo and colleagues,39 in which a TGF-ß-1 blocking antibody only partially reversed the endothelial growth suppression caused by exposure to pericyte-conditioned media, whereas the effect was completely ameliorated by heat treatment of the conditioned media. In concert with our data, this suggests that there are probably additional mediators of the Rho-dependent control of pericyte-endothelial cellular dynamics yet to be revealed.

Pericyte Dysregulation in Angiogenesis

Results of our in vitro studies may lead to a more complete understanding of the role that pericytes play in orchestrating microvascular proliferative disorders, whether tumor-dependent,40 diabetes-induced,41 or associated with aging.6 As we show here, pericyte signaling through a Rho GTPase-dependent pathway and its influence on endothelial cell growth arrest adds to a growing literature in support of pericytes?? contributory role in regulating capillary endothelial cell function. Indeed, although it has been proposed that pericyte apoptosis or loss is associated with several angiogenic processes,42 our findings strongly suggest that chronic alteration of pericyte Rho GTPase-dependent signaling may be sufficient for pathological angiogenesis to ensue. This underscores the importance of current work focusing on pericytes as novel drug targets in anti-angiogenic therapy.18-21

Pericytes in the Pathogenesis of Microvascular Tone Dysregulation

In addition to the role of disrupted endothelial control in vasoproliferative disease, microvascular signaling dysregulation is emerging as a causative factor in the pathogenesis of several nonproliferative vascular pathologies as well. Based on the initial understanding of Rho family GTPase signaling in the regulation of smooth muscle contractility,43 a key role for Rho signaling through Rho kinase in the vascular system has recently been elucidated in both physiological maintenance44 and pathophysiology.24 In particular, arterial Rho GTP-dependent signal transduction may play a pivotal role in the pathogenesis of essential hypertension (as recently reviewed by Lee et al23 ). Regulation of vascular tone is thought to be governed by the balance between active calcium-dependent contractility45 and nitric oxide-mediated relaxation46,47 in smooth muscle. Emerging clinical data from human patients indicates that inhibition of the Rho kinase pathway via intravenous infusion of the Rho kinase inhibitor fasudil can correct aberrations in peripheral vascular tone present in heart failure,48 coronary artery disease,49 and even in cigarette smoking50 with minimal tone alteration in normal controls. Our work suggests that an additional less well-explored component of the vascular pathologies accompanying essential hypertension may be linked to microvascular pericyte dysfunction or loss.

In the central nervous system, control of vascular tone is exquisitely sensitive to metabolic demands.51,52 Functional imaging studies in the cerebellum have shown that in vivo moment-to-moment local control of demand-induced hyperemia in response to increased synaptic activity is dependent on neuronal nitric-oxide synthase.53-56 Interestingly, inhibition of Rho kinase signaling by nitric oxide has been convincingly demonstrated in the regulation of extracranial arterial tone.47 Further, in situ hybridization and immunohistochemical evidence indicates robust expression of Rho GTPase and Rho kinase in cerebellar tissue,57 indicating that inhibition of Rho kinase signaling may be involved in cerebellar functional hyperemia. A similar potential role for Rho kinase has also been described in the hypertensive brainstem, including possible novel vasodilation-independent effects of Rho kinase activation in the regulation of the sympathetic nervous system.58 These results in concert suggest that perturbations in Rho-mediated signaling may underlie both vasogenic and neurogenic mechanisms of hypertension. Interestingly, early comparative studies on the cerebral microvasculature in hypertensive versus normotensive rats revealed increased pericyte investment of endothelial cells as well as loss of normal stress fiber distribution in pericytes associated with the hypertensive microvasculature both in situ and in cell culture.59,60

Parallel evidence from the retinal microvasculature supports the hypothesis that pericyte Rho kinase signaling may play a principle role in microvascular tone regulation. Careful dissection of smooth muscle contractile pathways in the hypertensive rat arterial system implicate activation of Rho kinase,61,62 dynamically balanced by countervailing Rho kinase-mediated release of nitric oxide from adjacent endothelial cells.63,64 In a pericyte-mediated parallel to this pathway in smooth muscle, calcium-dependent chloride channel activation mediates pericyte contractility in the retina,65-67 in which nitric oxide has similarly been shown to counterbalance ligand-mediated contraction by promoting pericyte relaxation.68,69 Interestingly, the retina is the most densely pericyte-invested vascular bed in the human body,70 lacking the precapillary smooth muscle sphincters that play a regulatory role in other vascular beds.71 Thus, pericyte Rho GTPase signaling through downstream Rho kinase effector pathways may play a microvascular bed-specific role parallel to that of smooth muscle regulation at the arteriolar level.

Our results indicate that pericyte contractility is Rho GTPase-dependent and Rho kinase-mediated. In concert with recent evidence that the pericyte is an autonomous regulator of capillary tone,72 these data imply that the Rho kinase-mediated regulation of vascular tone in specific capillary beds may in fact originate locally at the level of the capillary-associated pericyte, with subsequent upstream conduction to proximal arteriole-associated smooth muscle cells. This intriguing possibility suggests that some elements of vascular tone dysregulation previously attributed solely to vascular smooth muscle may in fact be pericyte-mediated and that further investigation into the regulation of microvascular tone and endothelial cell regulation may elucidate novel capillary level signaling mechanisms involving multiple perivascular cell types.

Acknowledgements

We thank Dr. Daniel Kalman, the Emory University School of Medicine, Atlanta. GA, for mutant Rho GTPase-containing adenoviruses; Dr. Deniz Toksoz, Tufts University School of Medicine, Boston, MA, for dominant-active Ras; Dr. Larry Feig, Tufts University School of Medicine, for dominant-active Rac1 and Cdc42; and Kathleen Riley and Lindsey Wolf for technical assistance.

【参考文献】
  Jain RK: Molecular regulation of vessel maturation. Nat Med 2003, 9:685-693

Hirschi KK, D??Amore PA: Pericytes in the microvasculature. Cardiovasc Res 1996, 32:687-698

Hungerford JE, Little CD: Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res 1999, 36:2-27

Darland DC, D??Amore PA: Cell-cell interactions in vascular development. Curr Top Dev Biol 2001, 52:107-149

Papetti M, Herman IM: Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol 2002, 282:C947-C970

Rattner A, Nathans J: Macular degeneration: recent advances and therapeutic opportunities. Nat Rev Neurosci 2006, 7:860-872

Fong DS, Aiello LP, Ferris FL, III, Klein R: Diabetic retinopathy. Diabetes Care 2004, 27:2540-2553

Li J, Zhang YP, Kirsner RS: Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix. Microsc Res Tech 2003, 60:107-114

Armulik A, Abramsson A, Betsholtz C: Endothelial/pericyte interactions. Circ Res 2005, 97:512-523

Rucker HK, Wynder HJ, Thomas WE: Cellular mechanisms of CNS pericytes. Brain Res Bull 2000, 51:363-369

Bergers G, Song S: The role of pericytes in blood-vessel formation and maintenance. Neuro-oncol 2005, 7:452-464

Betsholtz C, Lindblom P, Gerhardt H: Role of pericytes in vascular morphogenesis. EXS 2005, 94:115-125

Orlidge A, D??Amore PA: Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J Cell Biol 1987, 105:1455-1462

Sato Y, Rifkin DB: Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-ß1-like molecule by plasmin during co-culture. J Cell Biol 1989, 109:309-315

von Tell D, Armulik A, Betsholtz C: Pericytes and vascular stability. Exp Cell Res 2006, 312:623-629

Benjamin LE, Hemo I, Keshet E: A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 1998, 125:1591-1598

Hellström M, Gerhardt H, Kalen M, Li X, Eriksson U, Wolburg H, Betsholtz C: Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 2001, 153:543-553

Bagley RG, Rouleau C, Morgenbesser SD, Weber W, Cook BP, Shankara S, Madden SL, Teicher BA: Pericytes from human non-small cell lung carcinomas: an attractive target for anti-angiogenic therapy. Microvasc Res 2006, 71:163-174

Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D: Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003, 111:1287-1295

Pietras K, Hanahan D: A multitargeted, metronomic, and maximum-tolerated dose "chemo-switch" regimen is antiangiogenic, producing objective responses and survival benefit in a mouse model of cancer. J Clin Oncol 2005, 23:939-952

Shaheen RM, Tseng WW, Davis DW, Liu W, Reinmuth N, Vellagas R, Wieczorek AA, Ogura Y, McConkey DJ, Drazan KE, Bucana CD, McMahon G, Ellis LM: Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell survival mechanisms. Cancer Res 2001, 61:1464-1468

Hall A: Rho GTPases and the control of cell behaviour. Biochem Soc Trans 2005, 33:891-895

Lee DL, Webb RC, Jin L: Hypertension and RhoA/Rho-kinase signaling in the vasculature: highlights from the recent literature. Hypertension 2004, 44:796-799

Pacaud P, Sauzeau V, Loirand G: Rho proteins and vascular diseases. Arch Mal Coeur Vaiss 2005, 98:249-254

Kolyada AY, Riley KN, Herman IM: Rho GTPase signaling modulates cell shape and contractile phenotype in an isoactin-specific manner. Am J Physiol 2003, 285:C1116-C1121

Harris AK, Wild P, Stopak D: Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 1980, 208:177-179

Herman IM, D??Amore PA: Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol 1985, 101:43-52

Narumiya S, Ishizaki T, Uehata M: Use and properties of ROCK-specific inhibitor Y-27632. Methods Enzymol 2000, 325:273-284

Aebi U, Pollard TD: A glow discharge unit to render electron microscope grids and other surfaces hydrophilic. J Electron Microsc Tech 1987, 7:29-33

Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992, 89:5547-5551

Kalman D, Gomperts SN, Hardy S, Kitamura M, Bishop JM: Ras family GTPases control growth of astrocyte processes. Mol Biol Cell 1999, 10:1665-1683

Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S: Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997, 389:990-994

Papetti M, Shujath J, Riley KN, Herman IM: FGF-2 antagonizes the TGF-ß1-mediated induction of pericyte -smooth muscle actin expression: a role for myf-5 and Smad-mediated signaling pathways. Invest Ophthalmol Vis Sci 2003, 44:4994-5005

Sieczkiewicz GJ, Herman IM: TGF-ß1 signaling controls retinal pericyte contractile protein expression. Microvasc Res 2003, 66:190-196

Shih SC, Ju M, Liu N, Mo JR, Ney JJ, Smith LE: Transforming growth factor beta1 induction of vascular endothelial growth factor receptor 1: mechanism of pericyte-induced vascular survival in vivo. Proc Natl Acad Sci USA 2003, 100:15859-15864

Darland DC, D??Amore PA: TGF ß is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells. Angiogenesis 2001, 4:11-20

Sato Y, Tsuboi R, Lyons R, Moses H, Rifkin DB: Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system. J Cell Biol 1990, 111:757-763

Viñals F, Pouyssegur J: Transforming growth factor ß1 (TGF-ß1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF- signaling. Mol Cell Biol 2001, 21:7218-7230

Kondo T, Hosoya K, Hori S, Tomi M, Ohtsuki S, Terasaki T: PKC/MAPK signaling suppression by retinal pericyte conditioned medium prevents retinal endothelial cell proliferation. J Cell Physiol 2005, 203:378-386

Morikawa S, Baluk P, Kaidoh T, Haskell A, Jain RK, McDonald DM: Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am J Pathol 2002, 160:985-1000

Hammes HP: Pericytes and the pathogenesis of diabetic retinopathy. Horm Metab Res 2005, 37(Suppl 1):39-43

Gee MS, Procopio WN, Makonnen S, Feldman MD, Yeilding NM, Lee WM: Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy. Am J Pathol 2003, 162:183-193

Hirata K, Kikuchi A, Sasaki T, Kuroda S, Kaibuchi K, Matsuura Y, Seki H, Saida K, Takai Y: Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 1992, 267:8719-8722

Noma K, Oyama N, Liao JK: Physiological role of ROCKs in the cardiovascular system. Am J Physiol 2006, 290:C661-C668

Somlyo AP, Somlyo AV: Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003, 83:1325-1358

Carvajal JA, Germain AM, Huidobro-Toro JP, Weiner CP: Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol 2000, 184:409-420

Chitaley K, Webb RC: Nitric oxide induces dilation of rat aorta via inhibition of rho-kinase signaling. Hypertension 2002, 39:438-442

Kishi T, Hirooka Y, Masumoto A, Ito K, Kimura Y, Inokuchi K, Tagawa T, Shimokawa H, Takeshita A, Sunagawa K: Rho-kinase inhibitor improves increased vascular resistance and impaired vasodilation of the forearm in patients with heart failure. Circulation 2005, 111:2741-2747

Nohria A, Grunert ME, Rikitake Y, Noma K, Prsic A, Ganz P, Liao JK, Creager MA: Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease. Circ Res 2006, 99:1426-1432

Noma K, Higashi Y, Jitsuiki D, Hara K, Kimura M, Nakagawa K, Goto C, Oshima T, Yoshizumi M, Chayama K: Smoking activates Rho-kinase in smooth muscle cells of forearm vasculature in humans. Hypertension 2003, 41:1102-1105

Faraci FM, Heistad DD: Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev 1998, 78:53-97

Lauritzen M: Relationship of spikes, synaptic activity, and local changes of cerebral blood flow. J Cereb Blood Flow Metab 2001, 21:1367-1383

Akgören N, Fabricius M, Lauritzen M: Importance of nitric oxide for local increases of blood flow in rat cerebellar cortex during electrical stimulation. Proc Natl Acad Sci USA 1994, 91:5903-5907

Hayashi T, Katsumi Y, Mukai T, Inoue M, Nagahama Y, Oyanagi C, Yamauchi H, Shibasaki H, Fukuyama H: Neuronal nitric oxide has a role as a perfusion regulator and a synaptic modulator in cerebellum but not in neocortex during somatosensory stimulation??an animal PET study. Neurosci Res 2002, 44:155-165

Iadecola C, Li J, Ebner TJ, Xu X: Nitric oxide contributes to func-tional hyperemia in cerebellar cortex. Am J Physiol 1995, 268:R1153-R1162

Yang G, Chen G, Ebner TJ, Iadecola C: Nitric oxide is the predominant mediator of cerebellar hyperemia during somatosensory activation in rats. Am J Physiol 1999, 277:R1760-R1770

O??Kane EM, Stone TW, Morris BJ: Distribution of Rho family GTPases in the adult rat hippocampus and cerebellum. Brain Res Mol Brain Res 2003, 114:1-8

Ito K, Hirooka Y, Sakai K, Kishi T, Kaibuchi K, Shimokawa H, Takeshita A: Rho/Rho-kinase pathway in brain stem contributes to blood pressure regulation via sympathetic nervous system: possible involvement in neural mechanisms of hypertension. Circ Res 2003, 92:1337-1343

Herman IM, Jacobson S: In situ analysis of microvascular pericytes in hypertensive rat brains. Tissue Cell 1988, 20:1-12

Herman IM, Newcomb PM, Coughlin JE, Jacobson S: Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro. Tissue Cell 1987, 19:197-206

Moriki N, Ito M, Seko T, Kureishi Y, Okamoto R, Nakakuki T, Kongo M, Isaka N, Kaibuchi K, Nakano T: RhoA activation in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. Hypertens Res 2004, 27:263-270

Sagara Y, Hirooka Y, Nozoe M, Ito K, Kimura Y, Sunagawa K: Pressor response induced by central angiotensin II is mediated by activation of Rho/Rho-kinase pathway via AT1 receptors. J Hypertens 2007, 25:399-406

Löhn M, Steioff K, Bleich M, Busch AE, Ivashchenko Y: Inhibition of Rho-kinase stimulates nitric oxide-independent vasorelaxation. Eur J Pharmacol 2005, 507:179-186

Savoia C, Ebrahimian T, He Y, Gratton JP, Schiffrin EL, Touyz RM: Angiotensin II/AT2 receptor-induced vasodilation in stroke-prone spontaneously hypertensive rats involves nitric oxide and cGMP-dependent protein kinase. J Hypertens 2006, 24:2417-2422

Kawamura H, Kobayashi M, Li Q, Yamanishi S, Katsumura K, Minami M, Wu DM, Puro DG: Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina. J Physiol 2004, 561:671-683

Kawamura H, Oku H, Li Q, Sakagami K, Puro DG: Endothelin-induced changes in the physiology of retinal pericytes. Invest Ophthalmol Vis Sci 2002, 43:882-888

Wu DM, Kawamura H, Sakagami K, Kobayashi M, Puro DG: Cholinergic regulation of pericyte-containing retinal microvessels. Am J Physiol 2003, 284:H2083-H2090

Haefliger IO, Zschauer A, Anderson DR: Relaxation of retinal pericyte contractile tone through the nitric oxide-cyclic guanosine monophosphate pathway. Invest Ophthalmol Vis Sci 1994, 35:991-997

Sakagami K, Kawamura H, Wu DM, Puro DG: Nitric oxide/cGMP-induced inhibition of calcium and chloride currents in retinal pericytes. Microvasc Res 2001, 62:196-203

Shepro D, Morel NM: Pericyte physiology. FASEB J 1993, 7:1031-1038

Pannarale L, Onori P, Ripani M, Gaudio E: Precapillary patterns and perivascular cells in the retinal microvasculature. A scanning electron microscope study. J Anat 1996, 188:693-703

Peppiatt CM, Howarth C, Mobbs P, Attwell D: Bidirectional control of CNS capillary diameter by pericytes. Nature 2006, 443:700-704

作者单位：From the Department of Physiology,* Tufts University School of Medicine, Boston; The Kidney and Dialysis Research Laboratory, Caritas St. Elizabeth??s Medical Center, Boston; and the Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts