Collagen I Initiates Endothelial Cell Morphogenesis by Inducing Actin Polymerization through Suppression of Cyclic AMP and Protein Kinase A*

Mary C. Whelan and Donald R. SengerDagger

From the Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Received for publication, July 26, 2002, and in revised form, October 13, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Collagen I provokes endothelial cells to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords closely imitate the solid pre-capillary cords of embryonic angiogenesis, raising interesting questions about underlying mechanisms. Studies described here identify a critical mechanism beginning with collagen I ligation of integrins alpha 1beta 1 and alpha 2beta 1, followed by suppression of cyclic AMP and cyclic AMP (cAMP)-dependent protein kinase A, and marked induction of actin polymerization to form prominent stress fibers. In contrast to collagen I, laminin-1 neither suppressed cAMP nor protein kinase A activity nor induced actin polymerization or changes in cell shape. Moreover, fibroblasts did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape, thus indicating that collagen signaling, as observed in endothelial cells, does not extend to all cell types. Pharmacological elevation of cAMP blocked collagen-induced actin polymerization and formation of cords by endothelial cells; conversely, pharmacological suppression of either cAMP or protein kinase A induced actin polymerization. Collectively, these studies identify a previously unrecognized and critical mechanism, involving suppression of cAMP-dependent protein kinase A and induction of actin polymerization, through which collagen I drives endothelial cell organization into multicellular pre-capillary cords.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

During angiogenesis, proliferating endothelial cells (ECs)1 organize to form new three-dimensional capillary networks. This process has been studied extensively in the embryo, establishing that an early stage of angiogenesis involves transition of endothelial precursor cells to a spindle-shape morphology (1) in combination with alignment into solid, multicellular, pre-capillary, cord-like structures (2, 3). Moreover, these cord-like structures are interconnected to form a polygonal network (1, 4). Solid pre-capillary cords have also been observed during angiogenesis in the adult (5). These solid cords subsequently mature into tubes with hollow lumens for the transport of blood (1, 5).

Three-dimensional type I collagen provokes ECs in culture to undergo marked shape changes that closely imitate pre-capillary formation during embryonic angiogenesis. Within hours after addition of collagen I to confluent cultures, ECs partially retract and exhibit a spindle-shaped morphology, together with re-alignment to form solid cords organized in a polygonal pattern (6-10). Subsequently, over the course of several days, these structures mature to form tubes with hollow lumens through a process involving development and coalescence of intracellular vacuoles (11).

Consistent with the importance of collagens in regulating EC shape and multicellular organization into pre-capillary cords, there exists considerable evidence that interactions between collagens and ECs are highly relevant in vivo. For example, in the developing embryo, blood vessels arise from the organization of EC precursors within an extracellular matrix rich in collagens. Furthermore, during angiogenesis in the adult, ECs within existing blood vessels degrade basement membrane (12) and migrate and proliferate within connective tissue abundant in interstitial collagens (13). Consistent with the importance of collagen/EC interactions for angiogenesis, we reported previously that vascular endothelial growth factor (VEGF) induces microvascular ECs (MVECs) to express integrins alpha 1beta 1 and alpha 2beta 1 (14). Both of these integrins are key collagen receptors on MVECs, and antagonism of these two integrins inhibits dermal and tumor angiogenesis in vivo (14, 15), consistent with the importance of interactions between collagens and ECs. Also, recent analyses of genes expressed in human tumor endothelium demonstrated that ECs isolated from tumors express >10-fold more transcripts encoding collagens type I and III than ECs isolated from corresponding control tissue, indicating that tumor ECs express their own interstitial collagens (16). These findings suggest the interesting possibility that interstitial collagen expression by tumor ECs is conducive for angiogenesis. In support of this hypothesis, expression of collagen I by isolated EC clones in vitro closely correlates with spontaneous multicellular organization into cords (17, 18). Finally, neovascularization was inhibited in animal models both by proline analogues, which interfere with collagen triple helix assembly, and by beta -aminopropionitrile, which inhibits collagen cross-linking (19), indicating that collagens play a crucial role in angiogenesis.

Despite the abundant evidence indicating that interstitial collagens and their receptors are important for angiogenesis, little is known regarding the signaling events and mechanisms through which collagen regulates EC morphology and multicellular alignment into cords. Therefore, we designed experiments to identify key mechanisms involved. Our studies identify a series of critical steps beginning with collagen binding to integrins alpha 1beta 1 and alpha 2beta 1, followed by alpha 1beta 1- and alpha 2beta 1-mediated suppression of cyclic AMP and cyclic AMP-dependent protein kinase A (PKA), and induction of actin polymerization.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- Human dermal MVECs were isolated from neonatal foreskins and cultured as previously described (9). Fibroblasts were obtained as outgrowths from human neonatal foreskins, and they were maintained in Dulbecco's modified Eagle's medium containing 10% FCS. Cells were used at the fourth to seventh passage.

Purified recombinant human VEGF165 was obtained from the NCI Preclinical Repository, Biological Resources Branch, Frederick, MD. Sources of other reagents were as follows: rat tail collagen I (BD Biosciences, Bedford, MA); mouse laminin-1 (Invitrogen, Carlsbad, CA), camptothecin, forskolin, adenosine 3',5'-cyclic monophosphate 8-(4-chlorophenylthio)-sodium salt, 3-isobutyl-1-methylxanthine, pertussis toxin, MDL 12,330A, 2',3'-dideoxyadenosine, KT5720, and cell-permeable PKA inhibitor 14-22 amide (Calbiochem, San Diego, CA); DAPI, calcein AM, propidium iodide, latrunculin A, FITC-phalloidin, and Texas Red-DNase (Molecular Probes, Eugene, OR).

The following monoclonal antibodies (mAbs) were used: mouse anti-human integrin alpha 1 (clone FB12), mouse anti-human integrin a2 (clone P1E6), and rat anti-human alpha 6 (clone NKI-GoH3), all from Chemicon International, Temicula, CA; isotype IgG1 control mAb (anti-TNP, clone 107.3) was from Pharmingen, La Jolla, CA. For mAb clustering, either secondary goat anti-mouse or goat anti-rat Fc-specific antibodies (Abs) (Sigma, St. Louis, MO) were employed, as appropriate.

Stimulation of MVECs with Collagen I-- Cells were grown to confluence in a standard MVEC medium consisting of EBM-2 (Clonetics, San Diego, CA), 10% FCS, cyclic AMP (cAMP), and hydrocortisone (9). 24 h before use, the culture medium was removed and replaced with a simplified medium consisting of EBM-2 and 10% FCS to avoid any complications associated with the presence of exogenous cAMP. Acid-solubilized rat tail collagen I was neutralized and made isotonic according to the manufacturer's instructions and diluted in serum-free medium (EBM-2, Clonetics, San Diego, CA) to a concentration of 500 µg/ml unless indicated otherwise. Culture medium was gently removed from the cells and carefully replaced with the collagen-containing serum-free medium. Upon return of the cells to 37 °C, the collagen typically polymerized within 30 min. We found it important to minimize disturbance of the cells during the removal and replacement of the medium, because turbulent medium changes were observed to alter baseline cAMP. In addition to laminin-1 as a control, other controls for all of these experiments consisted of replicate wells to which we added the collagen solubilization vehicle (0.02 M acetic acid), neutralized and made isotonic identically to the collagen I solution and subsequently diluted identically to collagen in serum-free EBM, and replicate wells without a medium change. When following precautions for careful removal and replacement of the medium, we found no significant differences among controls regarding all parameters investigated.

Cell Survival Assays-- To detect apoptosis, MVECs were stained with FITC-Annexin-V (ApoTarget Kit from BioSource, Camarillo, CA), according to the manufacturer's instructions. Staining was measured with a SLT Spectrafluor fluorescent plate reader (excitation, 485 nm; emission, 535 nm). For positive controls, apoptosis was induced by incubating cells with camptothecin (1 µg/ml) for 6 h. Cell viability was measured with an established method involving the fluorescent calcein AM substrate (20, 21). Substrate fluorescence, indicative of live cells, was measured with a fluorescent plate reader (485-nm excitation, 535-nm emission). Cell death was assessed with propidium iodide staining (4 µM final concentration), which identifies loss of membrane integrity. Staining was measured with a fluorescent plate reader (590-nm excitation, 635-nm emission). Treatment of cells with Triton X-100 (0.01%) served as a positive control. To compare cell numbers, cells were fixed in buffered formalin, permeabilized with 0.01% Triton X-100, and stained with DAPI (400 ng/ml final concentration), which fluoresces upon binding to DNA. DAPI fluorescence was measured with a fluorescent plate reader (360-nm excitation, 465-nm emission).

Integrin Clustering and Blocking Experiments-- For experiments with integrin mAbs, expression of integrins alpha 1beta 1 and alpha 2beta 1 on the surface of MVECs was induced maximally by stimulating cells for 3 days with 20 ng/ml VEGF (14). To cluster integrins, integrin mAbs were incubated with cells in serum-free medium at a concentration of 50 µg/ml for 2 h, and secondary Fc-specific Ab (50 µg/ml) was added for 4 h. To block integrin ligation of collagen I, integrin mAbs were added at 10 µg/ml.

Assays for F-actin, G-actin, cAMP, and PKA-- For quantitative measurement of polymerized filamentous actin (F-actin) and globular actin (G-actin), cells on 96-well plates were fixed directly by adding an equal volume of phosphate-buffered formalin to the medium for 30 min. Plates then were washed by immersion in phosphate-buffered saline. F-actin was measured according to an established method (22) with the following modifications. Fixed cells were incubated for 45 min with FITC-phalloidin (200 mM) in phosphate-buffered saline containing 0.1% Triton X-100. Subsequently, the plates were washed five times, blotted, and analyzed with a spectrofluorometric plate reader (excitation, 485 nm; emission, 535 nm). Additional wells lacking cells were processed in parallel, and they served as background controls. G-actin was measured according to an established method (23). Plates were processed as described for F-actin assays except that fixed cells were incubated with Texas Red-DNase (300 nM). Fluorescent plate reader settings employed were as follows: excitation, 590 nm; emission, 630 nm.

For cAMP analyses (24-well plates), culture medium was removed and cells were extracted immediately with a -20 °C solution of 95% ethanol containing 500 µM 3-isobutyl-1-methylxanthine to inhibit phosphodiesterase activity. Samples were evaporated to dryness in a Speed-vac, and cAMP was measured with a cAMP enzyme-linked immunoassay kit (BioTechnologies, Stoughton, MA). PKA activity was measured with an assay kit (Calbiochem #539490) that measures incorporation of radiolabeled phosphate from [gamma -32P]ATP into a highly specific peptide substrate for PKA (LRRASLG). The extraction buffer for PKA also was supplemented with 500 µM 3-isobutyl-1-methylxanthine to inhibit phosphodiesterase activity after cell lysis.

Microscopy-- Fluorescence and phase-contrast images were collected with a Leica DC200 digital camera and associated software. For labeling of the actin cytoskeleton, cells were prepared and incubated with FITC-phalloidin as for the F-actin quantitative assays described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Through alpha 1beta 1 and alpha 2beta 1 Integrins, Collagen I Initiates a Morphogenetic Program in Dermal MVECs but Not Dermal Fibroblasts-- Conceivably, there are several strategies for studying collagen signaling in MVECs, including approaches that begin with cells in suspension. However, during the early stages of angiogenesis, adherent MVECs proliferate, invade, and re-organize in a three-dimensional matrix rich in interstitial collagens. Therefore, we chose a model system involving the addition of three-dimensional interstitial collagen I to adherent, confluent MVECs. This model better represents the conditions encountered by MVECs at the sprouting tips of blood vessels as they encounter the collagen-rich interstitial matrix.

As illustrated in Fig. 1A, adherent dermal MVECs responded to addition of collagen I with partial retraction and re-alignment into polygonal arrays of cord-like structures within 6 h. As described in the introduction, this morphogenetic process closely imitates an early organizational step in the formation of vascular plexuses during embryonic development in vivo (1, 4). In contrast to collagen I, equivalent concentrations of laminin-1 were without effect (not shown). Also, dermal fibroblasts did not respond detectably to collagen I, establishing that the morphogenetic response of MVECs to collagen I does not apply generally to all cell types (Fig. 1A).


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Fig. 1.   Through the alpha 1beta 1 and alpha 2beta 1 integrins, collagen I provokes morphogenesis of dermal MVECs but not dermal fibroblasts. A, representative microscopic fields illustrating that addition of 500 µg/ml collagen I to confluent human dermal MVECs provokes cell retraction and reorganization of the monolayer into cord-like structures within 6 h. By contrast, human dermal fibroblasts neither retract nor undergo morphogenesis. Bar = 100 µm. B, collagen-induced morphogenesis of MVECs is not attributable to apoptosis, as indicated by the lack of Annexin-V staining (for positive controls, cells were treated with camptothecin to induce apoptosis). Moreover, staining with calcein AM, which identifies live cells, indicated that cells were equally viable in the presence and absence of collagen I. Also, propidium iodide staining, which identifies cells with permeabilized membranes, did not distinguish between collagen-treated cells and controls; cell number, compared by quantifying fluorescence of DAPI, which binds DNA, was equivalent in both groups. Error bars = S.D. C, neither alpha 1-blocking mAb nor alpha 2-blocking mAb alone blocked collagen I-induced alignment of MVECs into cords; however, both mAbs in combination were highly effective. Bar = 100 µm.

Over more extensive time intervals, three-dimensional collagen has been found to impair EC survival (24). Therefore, we performed a series of control experiments to test the possibility that morphological changes we observed were related to induction of apoptosis or loss of cell viability or cell number. However, as shown in Fig. 1B, collagen I did not induce apoptosis or cell death in our experiments, as determined by Annexin-V staining and propidium iodide staining, respectively. Furthermore, collagen I did not reduce cell viability, as determined with the live cell marker, calcein AM (20, 21), and the cell number was not affected (Fig. 1B). Therefore, we conclude that neither induction of apoptosis nor loss of cell viability or cell number contributed to the morphological changes induced by collagen I in our experiments.

Next, we performed experiments with blocking mAbs to test the involvement of two prominent collagen receptors, the integrins alpha 1beta 1 and alpha 2beta 1, in mediating the shape changes induced in MVECs by collagen I. As shown in Fig. 1C, antagonism of either of these integrins individually failed to block collagen-induced changes in cell shape, but antagonism of both integrins in combination was highly effective. These findings implicate both alpha 1beta 1 or alpha 2beta 1 in mediating the shape changes provoked by collagen I. Similar to dermal MVECs, dermal fibroblasts have been shown previously to express both alpha 1beta 1 and alpha 2beta 1 (25); we confirmed the presence of these two integrins on our human dermal fibroblasts with immunohistochemistry (data not shown). Thus, the failure of dermal fibroblasts to respond to collagen I with shape changes similar to those observed with dermal MVECs is not attributable to the absence of either of these two integrins.

Collagen I Induces Actin Polymerization in MVECs but Not Fibroblasts, and Actin Polymerization Is Required for Formation of Cords-- To investigate the effects of collagen stimulation on the actin cytoskeleton, MVECs were stained with FITC-labeled phalloidin, which binds F-actin; and cells were examined with fluorescence microscopy. As shown in Fig. 2A, collagen I provoked a pronounced reorganization of F-actin in MVEC monolayers, and cellular alignment corresponded with alignment of prominent stress fibers. In addition, these experiments suggested that collagen I induced marked increases in F-actin. Therefore, F-actin was measured with a quantitative fluorescence assay. As shown in Fig. 2B, collagen I induced as much as a 300% increase in F-actin, and induction was dependent on collagen concentration. In addition, the marked increase in F-actin was paralleled by a corresponding decrease in G-actin, indicating that collagen drives substantial polymerization of the free actin pool. Collagen-induced increases in F-actin were routinely observed in numerous experiments with at least five different isolates of dermal MVECs. Some populations were more responsive than others, and cells at passage 5 or below were most responsive. Regardless, in all cases collagen I stimulation increased F-actin content by at least 100%. In contrast to collagen I and consistent with the failure of laminin-1 to provoke changes in MVEC shape, laminin-1 did not increase F-actin (Fig. 2B).


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Fig. 2.   Collagen-1 induces actin polymerization in MVECs. A, FITC-phalloidin staining of the actin cytoskeleton of MVECs indicates that collagen I provokes marked changes in F-actin within 4 h. Top panel: control; bottom panels: 4 h after addition of 500 µg/ml collagen I. Bars = 100 µm. As shown in the higher power view (bottom right), actin stress fibers align within cells forming cords (arrows). B, quantitative assays for F-actin and free G-actin establish that collagen I, but not laminin-1, stimulates a marked increase in actin polymerization by 4 h, resulting in substantial reduction in the free G-actin pool. Ab-mediated clustering of the alpha 1beta 1 or alpha 2beta 1 integrins, but not alpha 6 integrins, also provokes substantial actin polymerization in MVECs. Finally, in contrast to dermal MVECs, dermal fibroblasts do not respond to collagen I with detectable changes in F-actin. Error bars = S.D.

Because collagen I ligation of integrins alpha 1beta 1 and alpha 2beta 1 is required for collagen-induced MVEC alignment into cord-like structures (Fig. 1C), we also investigated the involvement of these integrins in regulating actin polymerization. Clustering of either the alpha 1beta 1 integrin or the alpha 2beta 1 integrin on MVECs with functional integrin mAbs stimulated increases in F-actin (Fig. 2B), thus implicating both of these integrins in mediating induction of actin polymerization. Consistent with findings that laminin-1 did not induce actin polymerization, mAb-mediated clustering of alpha 6 integrins, which bind laminins (26), did not increase F-actin.

In sharp contrast to dermal MVECs, fibroblasts did not respond to collagen I by increasing actin polymerization (Fig. 2B, bottom panel). These findings together with findings that collagen I does not provoke shape changes in fibroblasts (Fig. 1A) suggested the hypothesis that actin polymerization is critical to the mechanism by which collagen provokes MVEC retraction and re-organization into cord-like structures. Therefore, to test the significance of actin polymerization for cord formation by MVECs, we performed experiments with MVECs exposed to latrunculin A, a potent inhibitor of actin polymerization (27). As shown in Fig. 3A, latrunculin A blocked collagen-induced actin polymerization in MVECs, and it blocked collagen-induced changes in cell shape (Fig. 3B). Thus, these experiments establish that actin polymerization is critical to the mechanism by which collagen I initiates MVEC alignment into cords.


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Fig. 3.   Actin polymerization is required for collagen-induced formation of cords. A, latrunculin A (250 nM), which interferes with actin assembly by binding to G-actin, blocks collagen I-induced actin polymerization in MVECs (error bars = S.D.). B, collagen I was added to confluent MVEC cultures as in Fig. 1. Latrunculin A blocked collagen I-induced alignment of MVECs into cords. Bar = 100 µm.

Collagen I Provokes the Actin Polymerization Required for Cord Formation through Suppression of cAMP-- We observed that pharmacological elevation of intracellular cAMP consistently blocked collagen-induced cord formation by MVECs similar to a combination of alpha 1 and alpha 2 integrin mAbs and similar to latrunculin A (Fig. 4A). cAMP-elevating agents with similar blocking effects included forskolin (20-100 µM), which activates adenylate cyclase, and 3-isobutyl-1-methylxanthine (500 µM), which suppresses phosphodiesterase activity. Cell-permeable analogues of cAMP (100 µM) also blocked collagen-induced changes in MVEC shape. In particular, pharmacological elevation of cAMP with forskolin not only blocked collagen-induced cord formation, but it also blocked collagen-induced actin polymerization (Fig. 4B). Collectively, these findings suggested the possibility that the mechanism through which collagen I induces actin polymerization and stress fibers and thereby mediates MVEC organization into cords involves suppression of cAMP. Therefore, we measured intracellular cAMP in MVECs stimulated with collagen I. As shown in Fig. 4B, collagen I provoked a substantial decrease in cAMP; however, laminin-1, which neither provoked changes in cell shape nor induction of actin polymerization, did not suppress cAMP. Importantly, clustering of integrins alpha 1beta 1 and alpha 2beta 1 with mAbs individually (Fig. 4B) and together (not shown) also produced substantial decreases in intracellular cAMP, thus implicating both of these collagen receptors in mediating the action of collagen I. In contrast, mAb-clustering of alpha 6 integrins, which are laminin receptors, did not suppress cAMP. Also, collagen I did not regulate cAMP in dermal fibroblasts (Fig. 4B). Thus fibroblasts, in sharp contrast to MVECs, failed to respond detectably to collagen I with either suppression of cAMP, induction of actin polymerization, or changes in cell shape.


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Fig. 4.   Collagen-induced MVEC morphogenesis is regulated by cAMP. A, collagen I was added to confluent MVEC cultures as in Fig. 1, and pharmacological elevation of cAMP by forskolin (100 µM) blocked collagen-induced cellular alignment into cords. Bar = 100 µm. B, forskolin blocked collagen-induced actin polymerization (top panel). Furthermore, quantitative cAMP assays established that collagen I, but not laminin-1, suppressed cAMP in MVECs and that cAMP is suppressed by clustering of the alpha 1beta 1 and alpha 2beta 1 integrins but not by clustering of alpha 6 integrins (middle panels). Collagen I did not suppress cAMP in dermal fibroblasts, in marked contrast to MVECs. Error bars = S.D.

Collectively, the experiments described above established that collagen I suppresses cAMP in MVECs, and they suggested that the mechanism through which collagen I induces actin polymerization in MVECs involves suppression of cAMP. To test this hypothesis directly, we employed two synthetic antagonists of cAMP synthesis, 2',3'-dideoxyadenosine and MDL-12,330A, to achieve suppression of cAMP and thereby imitate the action of collagen I. Both of these compounds suppressed cAMP in MVECs, resulting in induction of F-actin (Fig. 5A) and actin stress fibers (Fig. 5B). Thus, these experiments established a functional connection between suppression of cAMP by collagen I and induction of actin polymerization in MVECs.


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Fig. 5.   Suppression of cAMP by collagen I is functionally linked to induction of actin polymerization in MVECs. A, adenylate cyclase inhibitors MDL 12,330A (2.5 mM) and 2',3'-dideoxyadenosine (2',3'-DDA) (90 µM) each suppressed cAMP in MVECs and induced F-actin within 4 h. Error bars = S.D. B, FITC-phalloidin staining indicated that F-actin induced by adenylate cyclase inhibitors organized into prominent stress fibers, similar to those observed with collagen I (Fig. 2A). Bar = 100 µm.

Collagen I Suppression of cAMP Results in Suppression of PKA Activity, and Suppression of PKA Induces Actin Polymerization in MVECs-- PKA activity is dependent upon cAMP, and consistent with our findings that collagen I suppressed cAMP (Fig. 4B), collagen I also provoked a marked decrease in PKA activity (Fig. 6A). Therefore, we tested the possibility that loss of PKA activity is a key consequence of cAMP suppression through which collagen I induces actin polymerization in MVECs. For these experiments we employed two specific inhibitors of PKA, KT5720 and a myristoylated synthetic peptide, representing the active portion of the natural PKA inhibitor PKI (28). Both of these inhibitors induced actin polymerization (Fig. 6A) and the appearance of prominent actin stress fibers (Fig. 6B). Thus, these experiments establish that collagen I stimulation of MVECs suppresses cAMP-dependent PKA activity in conjunction with suppression of cAMP. They also establish a functional connection between suppression of PKA activity, induction of actin polymerization, and induction of actin stress fibers.


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Fig. 6.   Suppression of cAMP by collagen I results in suppression of PKA in MVECs; suppression of PKA links suppression of cAMP to induction of actin polymerization. A, quantitative PKA assays established that collagen I suppresses cAMP-dependent PKA activity in MVECs, consistent with collagen suppression of cAMP. Specific PKA inhibitors KT5720 and a myristoylated PKI peptide sequence 14-22, representing the inhibitory domain of PKI, both induced F-actin thus establishing a functional connection between suppression of PKA activity and induction of actin polymerization. B, FITC-phalloidin staining indicated that F-actin induced by PKA inhibitors organized into prominent stress fibers, similar to those observed with collagen I (Fig. 2A) and adenylate cyclase inhibitors (Fig. 5B). Bar = 100 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Collagen I provokes MVECs in vitro to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords, which are organized in polygonal arrays, closely imitate the polygonal patterns of embryonic pre-capillary cords that precede the formation of mature blood vessels with lumens in vivo (1, 2, 4, 29). Experiments described here identify a previously unrecognized and critical mechanism through which collagen stimulates formation of cords. Specifically, these studies establish that collagen I drives MVECs to assemble into cords through ligation of integrins alpha 1beta 1 and alpha 2beta 1, followed by suppression of cAMP and suppression of cAMP-dependent PKA. Suppression of cAMP-dependent PKA induces actin polymerization and the formation of prominent F-actin stress fibers, which are required for cellular re-alignment into cords. Furthermore, collagen-induced actin polymerization in MVECs coincides with large reductions in G-actin, indicating that collagen I drives substantial polymerization of the free actin pool. Thus, by establishing a previously unrecognized and important connection between suppression of cAMP-dependent PKA and the induction of F-actin and MVEC alignment into cords, studies described here provide new understanding of the collagen-signaling mechanisms that regulate EC morphogenesis and multicellular organization.

Our finding that cAMP negatively regulates actin polymerization and EC assembly into pre-capillary cords implicates cAMP as a key gatekeeper of EC organization during angiogenesis. Importantly, cAMP has been shown to inhibit angiogenesis in vivo (30), however, the possibility that cAMP functions in regulating multicellular organization of ECs during angiogenesis has not been considered previously. Moreover, our findings suggest the possibility that elevation of cAMP and PKA may provide for clinical control of neovascularization by interfering with the early organizational stages of blood vessel formation.

Previously, the alpha 2beta 1 integrin alone had been implicated in mediating collagen-induced cord formation by umbilical vein ECs (8) The apparent discrepancy between those findings and our observations, which also implicate integrin alpha 1beta 1, is attributable to the fact that umbilical vein ECs do not express integrin alpha 1beta 1 and that both alpha 1beta 1 and alpha 2beta 1 are prominent collagen receptors on MVECs (14, 31). This distinction is important, because MVECs are derived from the small capillaries of the microvasculature, whereas umbilical vein ECs are derived from large veins. Thus, experiments described here indicate that both alpha 1beta 1 and alpha 2beta 1 mediate collagen-induced morphogenesis in microvascular endothelium, whereas such function may be limited to the alpha 2beta 1 integrin in large vessel endothelium, which does not express integrin alpha 1beta 1.

Although dermal fibroblasts express both the alpha 1beta 1 and alpha 2beta 1 integrins, these cells did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape. These findings illustrate marked differences in collagen signaling among cell types. Interestingly, such differences in collagen signaling may relate to the fact that fibroblasts normally reside within a collagen-rich matrix, whereas the MVECs of mature blood vessels are sequestered from interstitial collagen by basement membrane. Moreover, we found that laminin-1, a member of the laminin family of proteins which are major components of basement membranes (32, 33), did not suppress cAMP or initiate cord formation by MVECs, indicating that the responses of MVECs to collagen and laminin are distinctly different.

Stimulation of vascular smooth muscle cells with collagen I also has been reported to suppress intracellular cAMP (34). Pertussis toxin, which prevents inhibition of adenylate cyclase by heterotrimeric Gi proteins, blocked collagen suppression of cAMP in these cells, implicating Gi proteins in the mechanism (34). However, we found that collagen I suppressed cAMP substantially in MVECs treated with pertussis toxin (50 ng/ml, preincubated overnight) even though pertussis toxin elevated baseline cAMP in these cells (data not shown). Thus, we found no evidence that collagen I suppresses cAMP in MVECs through activation of Gi, and therefore it seems likely that collagen I regulates cAMP in MVECs differently than in smooth muscle cells. The possibility remains that collagen I suppresses cAMP in MVECs through activation of phosphodiesterases. Integrin alpha 6beta 4 has been shown to activate phosphodiesterase activity in carcinoma cells (35), and other integrins may also stimulate degradation of cAMP through this mechanism. Alternatively, collagen receptors may regulate cAMP metabolism through other mechanisms, consistent with the complexity of integrin signaling (36-39). Finally, data presented here raise the interesting question of how cAMP exerts negative control over actin polymerization in MVECs. The answer to this question may prove complex, because cAMP-dependent PKA regulates the activities of numerous proteins, including several that are implicated in cytoskeletal regulation (40, 41). Regardless, experiments described here identify a previously unrecognized regulatory role for cAMP in controlling actin polymerization and cord formation by microvascular endothelium.

In conclusion and summary, experiments described here identify an important mechanism through which collagen I provokes MVECs to form solid cord-like structures. These cords closely imitate the solid pre-capillary cords that appear during the early stages of angiogenesis in vivo. The signaling events through which collagen induces cord formation by MVECs begins with ligation of integrins alpha 1beta 1 and alpha 2beta 1, followed by suppression of cAMP and cAMP-dependent PKA. In turn, suppression of PKA induces actin polymerization, which is required for EC alignment and organization into cords. In contrast to dermal MVECs, dermal fibroblasts do not respond to collagen I with changes in cAMP, F-actin, or cell shape, underscoring important distinctions in collagen signaling among cell types. Although it has long been recognized that collagen I provokes ECs to initiate a morphogenetic program leading to the formation of pre-capillary cords, underlying signaling mechanisms had not been defined. Collectively, studies reported here identify a critical mechanism, and they identify cAMP as a key regulator of EC morphogenesis and multicellular organization.

    ACKNOWLEDGEMENTS

We thank Arthur Mercurio, Kathleen O'Connor, and Christopher Drake for helpful discussions.

    FOOTNOTES

* This work was supported by United States Public Health Services Grant CA77357 (to D. R. S.) from NCI, National Institutes of Health, and by the V. Kann Rasmussen Foundation.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.

Dagger To whom correspondence should be addressed: Dept. of Pathology, Research North, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-5766; Fax: 617-667-3591; E-mail: dsenger@caregroup.harvard.edu.

Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M207554200

    ABBREVIATIONS

The abbreviations used are: ECs, endothelial cells; MVECs, microvascular endothelial cells; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody; Ab, antibody; F-actin, filamentous actin; G-actin, globular actin; PKA, protein kinase A; PKI, natural protein kinase A inhibitor; DAPI, 4',6-diamidino-2-phenylindole; FCS, fetal calf serum; FITC, fluorescein isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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