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MT1-MMP is a membrane-tethered enzyme capable of remodeling extracellular matrix. MT1-MMP-deficient mice exhibit systematic defects during development, especially in craniofacial development characterized by retarded calvarial bone formation. Recently, we identified MT1-MMP as a critical positive modulator of FGF signaling during intramembranous ossification. MT1-MMP cleaves ADAM9 to protect FGFR2 from ectodomain shedding. Depletion of ADAM9 in MT1-MMP-deficient mice significantly rescued the calvarial defects via restoring FGF signaling. Interestingly, this regulatory mechanism seems to be highly tissue-specific, as defective FGF2-induced corneal angiogenesis in Mmp14−/−mice could not be rescued by removal of ADAM9. In addition, MT1-MMP also cleaves another ADAM family member, ADAM15. Our current findings not only present a novel regulatory mechanism for FGF signaling but also reveal a functional crosstalk between MMP and ADAM families. Better understanding of the interplay between ADAMs and MT1-MMP and its consequences for signaling pathways will provide new insights into therapeutic approaches for the management of developmental disorders and various diseases, such as cancer.

Cancer-associated retinopathy (CAR) is believed to be associated with autoimmune responses against retinal specific antigens.  However, CAR patients often show little evidence of immunological reactions at the cellular or molecular levels in their eyes. We have recently shown that tumor-derived VEGF and PlGF significantly remodel the retinal vasculature by ablation of pericytes and impair the blood-retinal barrier, leading to increased vascular leakage. Surprisingly, VEGFR1, but not VEGFR2, is the primary receptor that transduces signals in endothelial or mural cells to produce these vascular pathologies.  These results demonstrate that tumor-derived angiogenic factors significantly confer the development of CAR and anti-VEGF agents might be potentially used for the treatment of CAR.

Hepatocyte growth factor (HGF) has previously been reported to act as a hemangiogenic factor, as well as a mitogenic factor for a variety of tumor cells. Here, we demonstrate that HGF is a lymphangiogenic factor, which may contribute to lymphatic metastasis when overexpressed in tumors. In a mouse corneal lymphangiogenesis model, implantation of HGF induces sprouting and growth of new lymphatic vessel expressing the lymphatic vessel endothelial specific marker hyaluronan receptor-1 (Lyve-1). Unlike blood vessels, the Lyve-1–positive structures consist of blunt-ended vessels of large diameters that generally lack expression of CD31. The growth of HGF-induced lymphatic vessels can be partially blocked by a soluble VEGFR-3, suggesting that HGF may stimulate lymphatic vessel growth through an indirect mechanism. Consistent with this finding, the HGF receptor (c-Met) is only localized on corneal blood vessels but is absent on lymphatic vessels in a mouse corneal assay. In a transgenic mouse model that expresses HGF under the control of the whey acidic protein (WAP) gene promoter, transgenic females develop tumors in the mammary glands after several pregnancies. Interestingly, dilated Lyve-1–positive lymphatic vessels accumulate in the peritumoral area and occasionally penetrate into the tumor tissue. Our findings indicate that HGF may play a critical role in lymphangiogenesis and potentially contribute to lymphatic metastasis.

Hepatocyte growth factor (HGF) has previously been reported to act as a hemangiogenic factor, as well as a mitogenic factor for a variety of tumor cells. Here, we demonstrate that HGF is a lymphangiogenic factor, which may contribute to lymphatic metastasis when overexpressed in tumors. In a mouse corneal lymphangiogenesis model, implantation of HGF induces sprouting and growth of new lymphatic vessel expressing the lymphatic vessel endothelial specific marker hyaluronan receptor-1 (Lyve-1). Unlike blood vessels, the Lyve-1–positive structures consist of blunt-ended vessels of large diameters that generally lack expression of CD31. The growth of HGF-induced lymphatic vessels can be partially blocked by a soluble VEGFR-3, suggesting that HGF may stimulate lymphatic vessel growth through an indirect mechanism. Consistent with this finding, the HGF receptor (c-Met) is only localized on corneal blood vessels but is absent on lymphatic vessels in a mouse corneal assay. In a transgenic mouse model that expresses HGF under the control of the whey acidic protein (WAP) gene promoter, transgenic females develop tumors in the mammary glands after several pregnancies. Interestingly, dilated Lyve-1–positive lymphatic vessels accumulate in the peritumoral area and occasionally penetrate into the tumor tissue. Our findings indicate that HGF may play a critical role in lymphangiogenesis and potentially contribute to lymphatic metastasis.

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPCs) have been reported to incorporate into newly formed blood vessels under physiologic and pathologic conditions. However, it is unknown if CEPCs contribute to lymphangiogenesis. Here we show that in a corneal lymphangiogenesis model of irradiated mice reconstituted with enhanced green fluorescent protein (EGFP)-positive donor bone marrow cells, CEPCs are present in the newly formed lymphatic vessels. Depletion of bone marrow cells by irradiation remarkably suppressed lymphangiogenesis in corneas implanted with fibroblast growth factor-2 (FGF-2). Further, transplantation of isolated EGFP-positive/vascular endothelial growth factor receptor-3-positive (EGFP+/VEGFR-3+) or EGFP+/VEGFR-2+cell populations resulted in incorporation of EGFP+cells into the newly formed lymphatic vessels. EGFP+/CEPCs were also present in peritumoral lymphatic vessels of a fibrosarcoma. These data suggest that BM-derived CEPCs may play a role in “lymphvasculogenesis.”

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPCs) have been reported to incorporate into newly formed blood vessels under physiologic and pathologic conditions. However, it is unknown if CEPCs contribute to lymphangiogenesis. Here we show that in a corneal lymphangiogenesis model of irradiated mice reconstituted with enhanced green fluorescent protein (EGFP)-positive donor bone marrow cells, CEPCs are present in the newly formed lymphatic vessels. Depletion of bone marrow cells by irradiation remarkably suppressed lymphangiogenesis in corneas implanted with fibroblast growth factor-2 (FGF-2). Further, transplantation of isolated EGFP-positive/vascular endothelial growth factor receptor-3-positive (EGFP+/VEGFR-3+) or EGFP+/VEGFR-2+ cell populations resulted in incorporation of EGFP+ cells into the newly formed lymphatic vessels. EGFP+/CEPCs were also present in peritumoral lymphatic vessels of a fibrosarcoma. These data suggest that BM-derived CEPCs may play a role in “lymphvasculogenesis.”

The quiescent vascular system in the adult body represents the balanced net outcome of overproduction of endogenous angiogenesis inhibitors and reduced levels of angiogenic factors. While these inhibitors are expressed under physiological conditions, they are also generated in association with tumor growth. Angiostatin is such a specific angiogenesis inhibitor produced by tumors. It inhibits primary and metastatic tumor growth by blocking tumor angiogenesis. Encouraged by its potent anti-tumor activity, angiostatin is in clinical trials for cancer therapy. Angiostatin contains the first four triple loop structures, known as kringle domains, of plasminogen. The disulfide bond-linked kringle architectures are essential for the antiangiogenic activity of angiostatin. Based on this initial finding, recent work shows that kringle fragments of several other proteins also inhibit angiogenesis. Thus, kringle domains may provide a structural basis for identification of novel angiogenesis inhibitors. Surprisingly, most kringles only inhibit angiogenesis when cleaved as fragments from their parental proteins that lack antiangiogenic activity. These findings suggest that they are cryptic fragments hidden in large protein molecules. Thus, proteolytic processing plays a critical role in down regulation of angiogenesis. The kringle structure may provide the first example of a conserved architecture that specifically inhibits blood vessel growth. This review will focus on the structural and functional relationships of kringle domains in regulation of angiogenesis and tumor growth.

A newly discovered PDGF isoform, PDGF‐CC, is expressed in actively angiogenic tissues such as placenta, some embryonic tissues, and tumors. We test the possibility that PDGF‐CC promotes angiogenesis in vivo. The core domain (mature form) of human PDGF‐CC is sufficiently potent to stimulate neovascularization in the mouse cornea. The corneal angiogenic response induced by PDGF‐CC is robust although the area of neovascularization is smaller than those of FGF‐2‐and VEGF‐stimulated angiogenesis. Similarly, PDGF‐BB and PDGF‐AB induce angiogenic responses virtually indistinguishable from PDGF‐CCstimulated vessels. In contrast, PDGF‐AA displays only a weak angiogenic response in the mouse cornea. Although there was no significant difference in incorporation of mural cells to the newly formed blood vessels induced by PDGF‐BB and ‐CC, the percentage of mural cell positive vessels induced by PDGF‐AA was greater than those induced by FGF‐2, PDGF‐BB, and PDGF‐CC. In the developing chick embryo, PDGF‐CC induced branch sprouts from established blood vessels. In PDGF receptor‐transfected endothelial cells, PDGF‐CC activated the PDGF receptor alpha subunit (PDGFR‐a). PDGF‐CC, but not PDGF‐AA, was able to activate PDGFR‐p receptor in endothelial cells that coexpress both α and β forms of receptors. Thus, the PDGF‐CC‐mediated angiogenic response is most likely transduced by PDGF‐aa and ‐ap receptors. These data demonstrate that the PDGF family is a complex and important group of proangiogenic factors.—Cao, R., Bråkenhielm, E., Li, X., Pietras, K., Widenfalk, J, Östman, A., Eriksson, U., Cao, Y. Angiogenesis stimulated by PDGF‐CC, a novel member in the PDGF family, involves activation of PDGFR‐aa and ‐ap receptors. FASEB J. 16, 1575–1583 (2002)

The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin alpha4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin alpha4 chain in microvessel growth and, in the absence of other laminin alpha chains, in the composition of endothelial basement membranes.

ABSTRACTThe laminin α4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin α4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin α4 chain in microvessel growth and, in the absence of other laminin α chains, in the composition of endothelial basement membranes.

The laminin α4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin α4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin α4 chain in microvessel growth and, in the absence of other laminin α chains, in the composition of endothelial basement membranes.

Interleukin‐18 (IL‐18), also called interferon‐γ (IFN‐γ)‐inducing factor, has recently been characterized as a potent IFN‐γ‐inducing cytokine. We now report that IL‐18 is a novel antiangiogenic and antitumor cytokine. In vitro, IL‐18 specifically inhibits fibroblast growth factor‐2‐stimulated proliferation of capillary endothelial cells. In vivo, IL‐18 is sufficiently potent to suppress the fibroblast growth factor‐induced corneal neovascularization by systemic administration in mice. This cytokine also inhibits embryonic angiogenesis in the chick chorioallantoic membrane assay. Systemic and intralesional administrations of IL‐18 produce a significant suppression of the growth of murine T241 fibrosarcoma in syngeneic C57Bl6/J and immunodeficient SCID mice. The antitumor effect appears to be potent because an average of >75% inhibition of primary tumor growth was observed at a dose of 50 µg/kg/day. In cell culture, murine T241 fibrosarcoma cells are insensitive to recombinant IL‐18 at concentrations that significantly inhibit endothelial cell proliferation. Immunohistochemical studies of tumor tissues reveal hypovascularization of the IL‐18‐treated tumors. These results suggest that IL‐18 may participate in the regulation of a switch of tumor angiogenesis.—Cao, R., Farnebo, J., Kurimoto, M., Cao, Y. Interleukin‐18 acts as an angiogenesis and tumor suppressor. FASEB J.13, 2195–2202 (1999)

Although inflammation and metastasis are two well-known hallmarks of malignant disease, the relationship between inflammation and lymphatic metastasis is an unexplored research area. We recently elucidated a sophisticated mechanism by which TNFα-induced tumor inflammation conscripts macrophage-mediated VEGF-C-VEGFR3 signaling in lymphangiogenesis and metastasis.