Patricia D'Amore, Ph.D.

Regulation of Vascular Development and Growth

Research in the D'Amore laboratory focuses on the growth and maintenance of the microvasculature. This work is relevant to the retinal microangiopathies that occur as a complication of diabetes, including aspects of background diabetic retinopathy such as increased vascular permeability and pericyte dropout as well as the neovascularization that characterizes later stages of diabetic retinopathy. The studies are focused in two general areas: (1) cell-cell interactions and (2) the role and regulation of vascular endothelial growth factor isoforms.

1. Cell-Cell Interactions. The microvasculature consist of two cell types: the endothelial cell, which forms the capillary tube; and the pericyte, which is associated with the abluminal endothelial surface. Using a variety of co-culture systems, we have been examining the nature of the interactions between endothelial cells and pericytes, as well as the physiologic relevance of these interactions. Studies have been conducted to elucidate the mechanisms that regulate microvessel assembly. Co-culture of endothelial cells with mesenchymal pericyte precursors has revealed that endothelial cells can recruit undifferentiated mesenchymal cells by secreting PDGF B, which induces the directed migration as well as the proliferation of the precursors. Upon contact between the two cell types, the mesenchymal cells are induced to differentiated to a pericyte/smooth muscle lineage. This process is mediated, at least in part, by the activation of TGF-ß in the co-cultures. Current work is aimed at elucidating the molecular mechanisms, which underlie co-culture induced pericyte differentiation and the means by which pericytes mediate the stability of the microvessels.

2. Vascular Endothelial Growth Factor (VEGF). VEGF is a polypeptide growth factor that is an endothelial mitogen and angiogenic factor. We have previously shown that VEGF levels are increased in a non-human primate model of retinal ischemia-induced iris neovascularization. In addition, we showed that administration of soluble VEGF receptor (as a VEGF neutralizing reagent) can block ischemia-induced iris neovascularization. Molecular analysis in a tissue culture model revealed that VEGF gene expression is increased by hypoxia via the stabilization of VEGF mRNA. Current studies are aimed at investigating the role of VEGF in vascular development with particular emphasis on determining the distinct roles of the three different VEGF isoforms. To address this question we have used a genetic approach to generate mice that express single VEGF isoforms. Mice that express only VEGF 120 and not the two larger forms, VEGF 164 and VEGF 188, develop to term and then die rapidly as a result of a major deficiency in postnatal angiogenesis in various organs, including the lung and heart. Mice expressing only VEGF 164 or VEGF 188 have also been generated. Mice that express only VEGF188 are viable and fertile but display defects in retinal vascularization. Current studies are aimed at determining the mechanisms that underlie these differential phenotypes and determining if VEGF plays a role in the maintenance of the adult microvasculature.

References:

1. Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF- and heterotypic cell-cell interactions mediate the recruitment and differentiation of 10T1/2 cells to a smooth muscle cell fate. J Cell Biol, 1998; 141:805-814.

2. Hirschi KK, Rohovsky SA, Smith SR, Beck L, D’Amore PA. Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact. Circ Res., 1999; 84: 298-305.

3. Darland DC, D’Amore PA. Blood vessel maturation: Vascular development comes of age. Journal of Clinical Investigation, 1999; 103: 157-158.

4. Carmeliet P, Ng Y-S, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard J-C, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, D’Amore PA, Shima DT. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the VEGF ₁₆₄and VEGF ₁₈₈isoforms. Nature Medicine, 1999, 5:495-502.

5. Grosskreutz, CL, Anand-Apte, B, Duplaa, C, Quinn, TP, Terman, BI, Zetter, B, D’Amore, PA. Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. Microvascular Research, 1999, 58:128-136.

6. Hartnett, ME, Garcia, CM, D’Amore, PA. Release of bFGF, an endothelial cell survival factor, by osmotic shock. Investigative Ophthalmology & Visual Science, 1999, 40:2945-2951.

7. D’Amore, PA. Kissing Cousins-evidence for a common vascular cell precursor. Nature Medicine, 2000, 6:1323-1324.

8. Ng, Y-S, Rohan, R, Sunday, ME, deMello, DE, D’Amore, PA. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev Dyn, 2001, 220:112-121.

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

10. Nguyen, LL, D’Amore, PA. Cellular interactions in vascular growth and differentiation. International Review of Cytology, 2001, 204:1-48.

11. Stalmans, I, Ng, Y-S, Rohan, R, Fruttiger, M, Bouche, A, Yuce, A, Fujisawa, H, Hermans, B, Shani, M, Jansen, S, Hicklin, D, Anderson, DJ, Gardiner, T, Hammes, H-P, Moons, L,

12. Dewerchin, M, Collen, D, Carmeliet, P, and D’Amore, PA. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J Clin Invest, 2002, 109:327-336.

13. D’Amore, PA, Ng, Y-S. Won’t you be my neighbor? Local induction of arteriogenesis. Cell, 2002; 110:289-292 (Invited commentary).

14. Ramsauer, M, D’Amore, PA. Getting Tie(2)d up in angiogenesis. J Clin Invest, 2002; 110:1615-1617 (Invited commentary).

15. Ishida, S, Usui, T, Yamashiro, K, Kaji, Y, Amano, S, Ogura, Y, Hida, T, Oguchi, Y, Ambati, J, Ng, Y-S, D’Amore, PA, Shima, DT, Adamis, AP. VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced neovascularization, The Journal of Experimental Medicine, 2003;198:483-9

16. Darland DC, Massingham LJ, Smith SR, Piek E, Saint-Geniez M, D'Amore PA. Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev Biol. 2003;264:275-88.

17. Beck LH Jr, Goodwin AM, D'Amore PA. Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo.Differentiation. 2004;72:162-70


	Patricia D'Amore, Ph.D. Regulation of Vascular Development and Growth Research in the D'Amore laboratory focuses on the growth and maintenance of the microvasculature. This work is relevant to the retinal microangiopathies that occur as a complication of diabetes, including aspects of background diabetic retinopathy such as increased vascular permeability and pericyte dropout as well as the neovascularization that characterizes later stages of diabetic retinopathy. The studies are focused in two general areas: (1) cell-cell interactions and (2) the role and regulation of vascular endothelial growth factor isoforms. 1. Cell-Cell Interactions. The microvasculature consist of two cell types: the endothelial cell, which forms the capillary tube; and the pericyte, which is associated with the abluminal endothelial surface. Using a variety of co-culture systems, we have been examining the nature of the interactions between endothelial cells and pericytes, as well as the physiologic relevance of these interactions. Studies have been conducted to elucidate the mechanisms that regulate microvessel assembly. Co-culture of endothelial cells with mesenchymal pericyte precursors has revealed that endothelial cells can recruit undifferentiated mesenchymal cells by secreting PDGF B, which induces the directed migration as well as the proliferation of the precursors. Upon contact between the two cell types, the mesenchymal cells are induced to differentiated to a pericyte/smooth muscle lineage. This process is mediated, at least in part, by the activation of TGF-ß in the co-cultures. Current work is aimed at elucidating the molecular mechanisms, which underlie co-culture induced pericyte differentiation and the means by which pericytes mediate the stability of the microvessels. 2. Vascular Endothelial Growth Factor (VEGF). VEGF is a polypeptide growth factor that is an endothelial mitogen and angiogenic factor. We have previously shown that VEGF levels are increased in a non-human primate model of retinal ischemia-induced iris neovascularization. In addition, we showed that administration of soluble VEGF receptor (as a VEGF neutralizing reagent) can block ischemia-induced iris neovascularization. Molecular analysis in a tissue culture model revealed that VEGF gene expression is increased by hypoxia via the stabilization of VEGF mRNA. Current studies are aimed at investigating the role of VEGF in vascular development with particular emphasis on determining the distinct roles of the three different VEGF isoforms. To address this question we have used a genetic approach to generate mice that express single VEGF isoforms. Mice that express only VEGF 120 and not the two larger forms, VEGF 164 and VEGF 188, develop to term and then die rapidly as a result of a major deficiency in postnatal angiogenesis in various organs, including the lung and heart. Mice expressing only VEGF 164 or VEGF 188 have also been generated. Mice that express only VEGF188 are viable and fertile but display defects in retinal vascularization. Current studies are aimed at determining the mechanisms that underlie these differential phenotypes and determining if VEGF plays a role in the maintenance of the adult microvasculature. References: 1. Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF- and heterotypic cell-cell interactions mediate the recruitment and differentiation of 10T1/2 cells to a smooth muscle cell fate. J Cell Biol, 1998; 141:805-814. 2. Hirschi KK, Rohovsky SA, Smith SR, Beck L, D’Amore PA. Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact. Circ Res., 1999; 84: 298-305. 3. Darland DC, D’Amore PA. Blood vessel maturation: Vascular development comes of age. Journal of Clinical Investigation, 1999; 103: 157-158. 4. Carmeliet P, Ng Y-S, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard J-C, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, D’Amore PA, Shima DT. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the VEGF ₁₆₄and VEGF ₁₈₈isoforms. Nature Medicine, 1999, 5:495-502. 5. Grosskreutz, CL, Anand-Apte, B, Duplaa, C, Quinn, TP, Terman, BI, Zetter, B, D’Amore, PA. Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. Microvascular Research, 1999, 58:128-136. 6. Hartnett, ME, Garcia, CM, D’Amore, PA. Release of bFGF, an endothelial cell survival factor, by osmotic shock. Investigative Ophthalmology & Visual Science, 1999, 40:2945-2951. 7. D’Amore, PA. Kissing Cousins-evidence for a common vascular cell precursor. Nature Medicine, 2000, 6:1323-1324. 8. Ng, Y-S, Rohan, R, Sunday, ME, deMello, DE, D’Amore, PA. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev Dyn, 2001, 220:112-121. 9. Darland, DC, D’Amore, PA. TGFb is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells. Angiogenesis, 2001 4:11-20. 10. Nguyen, LL, D’Amore, PA. Cellular interactions in vascular growth and differentiation. International Review of Cytology, 2001, 204:1-48. 11. Stalmans, I, Ng, Y-S, Rohan, R, Fruttiger, M, Bouche, A, Yuce, A, Fujisawa, H, Hermans, B, Shani, M, Jansen, S, Hicklin, D, Anderson, DJ, Gardiner, T, Hammes, H-P, Moons, L, 12. Dewerchin, M, Collen, D, Carmeliet, P, and D’Amore, PA. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J Clin Invest, 2002, 109:327-336. 13. D’Amore, PA, Ng, Y-S. Won’t you be my neighbor? Local induction of arteriogenesis. Cell, 2002; 110:289-292 (Invited commentary). 14. Ramsauer, M, D’Amore, PA. Getting Tie(2)d up in angiogenesis. J Clin Invest, 2002; 110:1615-1617 (Invited commentary). 15. Ishida, S, Usui, T, Yamashiro, K, Kaji, Y, Amano, S, Ogura, Y, Hida, T, Oguchi, Y, Ambati, J, Ng, Y-S, D’Amore, PA, Shima, DT, Adamis, AP. VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced neovascularization, The Journal of Experimental Medicine, 2003;198:483-9 16. Darland DC, Massingham LJ, Smith SR, Piek E, Saint-Geniez M, D'Amore PA. Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev Biol. 2003;264:275-88. 17. Beck LH Jr, Goodwin AM, D'Amore PA. Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo.Differentiation. 2004;72:162-70