HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 20/6/717 05-5131fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ballard, V. L. T. Articles by Edelberg, J. M. Search for Related Content PubMed PubMed Citation Articles by Ballard, V. L. T. Articles by Edelberg, J. M.

Vascular tenascin-C regulates cardiac endothelial phenotype and neovascularization

Victoria L. T. Ballard^*, Arti Sharma^*, Inga Duignan^*, Jacquelyne M. Holm^*, Andrew Chin^*, Ruby Choi^*, Katherine A. Hajjar, Shing-Chiu Wong^* and Jay M. Edelberg^*,,1

Department of
^* Medicine and Department of
Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York, USA

¹Correspondence: Dept. of Medicine, Div. of Cardiology, Weill Medical College of Cornell University, 520 East 70th St., New York, NY 10021, USA. E-mail: jme2002{at}med.cornell.edu

SPECIFIC AIMS

Cardiac repair after ischemia and thrombosis involves contributions from both local endothelial cells and bone marrow-derived endothelial progenitor cells (EPCs) for blood vessel remodeling and neovascularization. A number of microenvironmental signals regulate the activity of both of these cell types in response to vascular injury, suggesting that similar mechanisms regulate angiogenesis in the vascular beds of both the heart and bone marrow. Therefore, we sought to identify epitopes expressed in the cardiac and bone marrow vasculature that may regulate such parallel mechanisms of postnatal neovascularization.

PRINCIPAL FINDINGS

1. Tenascin-C is expressed in the cardiac vasculature
An in vivo phage biopan was performed to identify epitopes that may regulate angiogenesis in both the heart and bone marrow. A phage clone, designated R3Y32, with homology to 8-integrin was found to have 10- and 6-fold higher levels of R3Y32 binding in young compared with old murine hearts and bone marrow, respectively. This suggested that binding partners for 8-integrin are preferentially expressed in the vasculature of the young bone marrow and heart. Immunohistochemical analysis of 8-integrin binding partners demonstrated that tenascin-C is localized to the cardiac vasculature and co-localizes with sites of phage binding. While tenascin-C is known to be up-regulated in the heart at sites of myocardial infarction, we have established for the first time that it is expressed in the healthy, intact heart at endovascular locations. Immunohistochemistry confirmed that tenascin-C is also localized to the bone marrow vasculature. Based on these findings, tenascin-C was further analyzed for potential roles in the promotion of pro-angiogenic pathways (Fig. 1 ).

View larger version (72K): [in this window] [in a new window]   Figure 1. R3Y32 binding in young and old bone marrow and hearts. A) Phage R3Y32 variable region epitope with sequence homology to extracellular domain of 8-integrin. Quantification of R3Y32 binding in young and old bone marrow and hearts (n=4 per group). *P<0.001. B) Co-immunostaining of tenascin-C (red) and phage coat protein pIII (green) in sections of the young rodent heart, demonstrating binding of R3Y32 to areas of tenascin-C expression in cardiac venules and microvessels. Insets illustrate separate signals for tenascin-C and pIII immunostains and negative control with no primary antibodies. C) Tenascin-C expression in a venule of the heart. D) Tenascin-C and DAPI co-stain demonstrate luminal expression of tenascin-C in an arteriole. E) Tenascin-C in bone marrow vasculature (arrowheads). Inset, negative control. F) Co-immunostaining of fibronectin (red) and phage coat protein pIII (green) in sections of the young rodent heart (arrows). Insets, separate signals for fibronectin and pIII immunostains. G) Fibronectin expression in a venule of the heart (arrows). B, D–G) Scale bars, 50 µm; C and inset in E) 100 µm.

2. Tenascin-C exerts a dynamic anti-adhesive effect on cardiac microvascular endothelial cells
Tenascin-C has been shown to exert both adhesive and anti-adhesive effects, and pro- and anti-migratory effects depending on the cell type with which it interacts. In vitro analysis demonstrated that tenascin-C exerts a temporally dynamic effect on cardiac microvascular endothelial cells (CMECs): in contrast to CMECs cultured on collagen or fibronectin, which spread and rapidly attach to the substrate within 3h of plating, CMECs cultured on tenascin-C remain highly spherical and are anti-adhesive at early time points. By 24 h, cells on tenascin-C are well attached and spread. We hypothesized that the initial disadhesive actions of tenascin-C on CMECs might function as an angiogenic inducer to promote the migratory capacity of CMECs. Using a 3-dimensional collagen gel assay system, we observed that CMECs cultured on tenascin-C for 3h displayed enhanced migratory capacity compared with those cultured on collagen. This migratory activity was further enhanced in response to the angiogenic growth factors PDGF-AB and VEGF-A.

3. Tenascin-C is associated with sites of cardiac angiogenic activity
We have previously demonstrated that PDGF signaling is important for postnatal cardiac remodeling, promoting angiogenesis mediated by a subpopulation of PDGFR-positive endothelial cells and bone marrow-derived EPCs. Given that PDGF is also known to induce tenascin-C expression in other vascular cell types, we hypothesized that tenascin-C may act as a downstream component of PDGF-mediated angiogenic mechanisms. In vitro RT-PCR analysis of tenascin-C expression in CMECs cultured with PDGF revealed an up-regulation of tenascin-C mRNA, peaking at 6 h of PDGF-AB treatment. Twenty-four h after intramyocardial injection of PDGF-AB, PDGFR-positive cells were localized at sites of tenascin-C protein expression in the cardiac vasculature. Together these data suggest that tenascin-C can act downstream of PDGF-AB and may regulate both local endothelial cell function as well as EPC incorporation. To further examine the association of tenascin-C with EPCs in the heart, genetically tagged ROSA-26 (ß-gal+) bone marrow cells were injected systemically into irradiated young mice, followed 1 month later by intramyocardial administration of PDGF-AB. This resulted in donor-derived (ß-gal+) cell recruitment to the heart within 24 h of PDGF-AB treatment, with the majority of cells incorporating at sites of tenascin-C protein expression. Thus, tenascin-C is associated with sites of EPC integration into the cardiac vasculature.

4. Tenascin-C is necessary for thrombus vascularization
Since tenascin-C appears to regulate proangiogenic mechanisms, we hypothesized that this protein would be present at sites of angiogenesis in human pathophysiological conditions. Based on the observed endovascular patterning of tenascin-C and the reported role of EPCs in channel formation in intravascular thrombi, we examined human cardiac thrombi extracted by percutaneous intracoronary thrombectomy from individuals with acute coronary syndromes. Immunostains revealed that tenascin-C is expressed at sites of intrathrombi channels and is colocalized with the endothelial/EPC marker Tie-2. This is the first identification of tenascin-C in coronary thrombi and its localization in the vasculature supports a role for this protein in recanalization of fibrin clots via neovascularization.

To determine whether tenascin-C is necessary for fibrin vascularization, we employed a mouse model of cardiac angiogenesis. Wild-type neonatal hearts were transplanted into the pinnae of young wild-type or tenascin-C^–/– mice. 3 days after transplantation, the periallograft region of wild-type host mice revealed formation of a fibrin clot. These thrombi were infiltrated by vascular channels that were positive for tenascin-C protein and were often associated with Tie-2-positive cells. Analysis of tenascin-C^–/– host mice at 3 days also showed significant fibrin deposition. However, no vascular channels were present in these clots. These data therefore confirm that tenascin-C is essential for fibrin vascularization (Fig. 2 ).

View larger version (42K): [in this window] [in a new window]   Figure 2. Tenascin-C–/– mice fail to vascularize cardiac thrombi. A) Model of cardiac allograft transplantation. Neonatal wild-type hearts are inserted into the pinnae of young wild-type or tenascin-C–/– mice. Seven days later, viability is scored based on visual assessment of healthy cardiac allograft and lack of thrombosis (B, C), as well as EKG activity of the allograft (C). 3 days after transplantation, Giemsa-stained sections of the pinnae of wild-type mice display fibrin vascularization (E, F). Immunostaining reveals that these vessels are positive for tenascin-C (brown signal in upper and lower left insets, G; green signal in right inset, G) and are associated with Tie-2-positive (red signal in right inset, G) cells. In contrast, sections of allografts in tenascin-C–/– mice display evidence of thrombus formation, but with no associated vascularization (F, G). E, H) Yellow arrows, transplanted heart tissue; black arrows, fibrin of thrombus; F, I) *regions of each section shown at higher magnification; G) yellow arrows, DAB-positive tenascin-C signal. E, F) Scale bars, 250 µm; F, I) 100 µm.

5. Tenascin-C is essential for cardiac angiogenesis
Given the deficiency in fibrin neovascularization observed 3 days after cardiac allograft transplantation, we hypothesized that a lack of tenascin-C would inhibit subsequent vascularization of the cardiac allografts. Analysis of cardiac allografts in wild-type mice 7 days after transplantation revealed that the fibrin had resolved and the transplanted cardiac tissue itself was vascularized. By contrast, the allografts transplanted into tenascin-C^–/– mice were not viable and significant thrombosis was still apparent at the engraftment site. Thus, tenascin-C is an essential component of cardiac neovascularization.

Work from our group and others has shown that bone marrow-derived EPCs contribute to cardiac angiogenesis. Indeed, we have demonstrated that the down-regulation of angiogenic mechanisms in the aging animal can be restored with transplantation of bone marrow cells from young, but not old, mice. Based on our findings that tenascin-C is expressed in the bone marrow vasculature and tenascin-C^–/– mice are known to have defects in bone marrow cell function, we sought to determine whether the actions of tenascin-C on the bone marrow cells are also essential for cardiac angiogenesis. We performed bone marrow transplantation from young wild-type or tenascin-C^–/– mice into aging hosts, prior to cardiac allograft transplantation. Young wild-type bone marrow cells transplanted into intact aging hosts were able to restore angiogenic function that is down-regulated with age. Transplantation of bone marrow from young tenascin-C^–/– mice, however, failed to vascularize the cardiac allografts, demonstrating that tenascin-C is essential for bone marrow cell-mediated mechanisms of post-natal neovascularization. While donor tenascin-C^–/– bone marrow cells were able to colonize the host bone marrow, the paucity of these cells in the periallograft region demonstrates the importance of tenascin-C in bone marrow cell-mediated cardiac angiogenic function.

CONCLUSIONS AND SIGNIFICANCE

Endothelial dysfunction is associated with increased risk of cardiovascular disease. Understanding the factors that are important to maintain or restore angiogenic/vasculogenic mechanisms is necessary for the development of therapies to treat, and possibly prevent, cardiovascular disease. We have identified tenascin-C as a regulator of such mechanisms. Tenascin-C has been implicated in wound healing and angiogenesis in many tissues and disease states but its role in the heart has not previously been defined. Our findings represent the first demonstration that tenascin-C is expressed in the cardiac vasculature. Importantly, it is found at sites of activated endothelial cells and regions of EPC incorporation. Mechanistically, we have established that tenascin-C promotes an early antiadhesive, promigratory phenotype upon interaction with CMECs. This is consistent with in vivo expression at sites of fibrin canalization in both humans and in our experimental model of thrombosis. Together, these data strongly suggest a role for tenascin-C in local vascular remodeling.

This role is further confirmed by our cardiac allograft transplantation studies which, for the first time, demonstrate that tenascin-C is essential for cardiac angiogenesis. Furthermore, we have demonstrated that tenascin-C is essential for bone marrow-mediated angiogenic function. Thus, the interactions of tenascin-C with endothelial cells and EPCs may represent a novel approach for enhancing mechanisms of vascular repair that are known to be down-regulated with age and in pathological conditions.

View larger version (27K): [in this window] [in a new window]   Figure 3. We propose that tenascin-C expression is up-regulated in the cardiac vasculature by proangiogenic cytokines, such as PDGF released by platelets, in response to vascular damage. As a result, local endothelial cells are activated and EPCs are recruited to the site of tenascin-C expression. This angiogenic induction, mediated by tenascin-C promotes subsequent migration of endothelial cells and cardiac neovascularization.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5131fje;

This article has been cited by other articles:

				M. Degen, F. Brellier, R. Kain, C. Ruiz, L. Terracciano, G. Orend, and R. Chiquet-Ehrismann Tenascin-W Is a Novel Marker for Activated Tumor Stroma in Low-grade Human Breast Cancer and Influences Cell Behavior Cancer Res., October 1, 2007; 67(19): 9169 - 9179. [Abstract] [Full Text] [PDF]

				V. L.T. Ballard and J. M. Edelberg Stem Cells and the Regeneration of the Aging Cardiovascular System Circ. Res., April 27, 2007; 100(8): 1116 - 1127. [Abstract] [Full Text] [PDF]

				V. L. T. Ballard, J. M. Holm, and J. M. Edelberg Quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling Physiol Genomics, September 14, 2006; 26(3): 202 - 208. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 20/6/717 05-5131fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ballard, V. L. T. Articles by Edelberg, J. M. Search for Related Content PubMed PubMed Citation Articles by Ballard, V. L. T. Articles by Edelberg, J. M.