John J. Castellot

Low Molecular Weight Inhibitors in Corneal Ulcerationa

RICHARD E. GALARDYP MARIE E. CASSABONNE, C A R L A ” E GIESE, JAMES H. GILBERT, FRANCE LAPIERRE, HENRY LOPEZ, MARY E. SCHAEFER, ROBERT STACK, MICHAEL SULLIVAN, BRENT SUMMERS, ROB TRESSLER, DAVE TYRRELL, AND JENNIFER WEEC; SCOTT D. ALLEN AND JOHN J. CASTELLO@; JOHN P. BARLETTA AND GREGORY S. SCHULTZe; LEONARD0 A. FERNANDEZf; SUSAN FISHER AND TIAN-YI CUF; HARALD G. FOELLMERh; DAMIAN GROBELNY’; AND WALTER M. HOLLERANJ

Impaired Clearance of Apoptotic Cells Promotes Synergy between Atherogenesis and Autoimmune Disease

To clarify the link between autoimmune disease and hypercholesterolemia, we created the gld.apoE −/− mouse as a model of accelerated atherosclerosis. Atherosclerotic lesion area was significantly increased in gld.apoE −/− mice compared with apoE −/− mice. gld.apoE −/− mice also displayed increases in lymphadenopathy, splenomegaly, and autoantibodies compared with gld mice, and these effects were exacerbated by high cholesterol diet. gld.apoE −/− mice exhibited higher levels of apoptotic cells, yet a reduced frequency of engulfed apoptotic nuclei within macrophages. Infusion of lysophosphatidylcholine, a component of oxidized low density lipoprotein, markedly decreased apoptotic cell clearance in gld mice, indicating that hypercholesterolemia promotes autoimmune disease in this background. These data suggest that defects in apoptotic cell clearance promote synergy between atherosclerotic and autoimmune diseases.

1-Butyryl-Glycerol: A novel angiogenesis factor secreted by differentiating adipocytes

Differentiation of adipocytes is accompanied by secretion of molecules stimulating angiogenesis in vivo and endothelial cell growth and motility in vitro. We demonstrate that the angiogenic and motility-stimulating activities secreted by adipocytes are separable from the endothelial cell mitogenic activity by fractionation of adipocyte-conditioned medium. The major differentiation-dependent angiogenic molecule was purified and identified by GCMS as 1-butyryl-glycerol (monobutyrin). Monobutyrin levels increase at least 200-fold during adipocyte differentiation and represent a major fraction of the total angiogenic activity. Synthetic monobutyrin shows the same spectrum of biological activities as the adipocyte-derived factor: stimulation of angiogenesis in vivo and microvascular endothelial cell motility in vitro, with no effect on endothelial cell proliferation. Angiogenesis is stimulated at doses as low as 20 pg when tested in the chick chorioallantoic membrane assay. These results strongly suggest that monobutyrin is a key regulatory molecule in an angiogenic process linked to normal cellular and tissue development.

Selective Activation of Peroxisome Proliferator–Activated Receptor (PPAR)α and PPARγ Induces Neoangiogenesis Through a Vascular Endothelial Growth Factor–Dependent Mechanism

OBJECTIVE—Peroxisome proliferator–activated receptors (PPARs) are therapeutic targets for fibrates and thiazolidinediones, which are commonly used to ameliorate hyperlipidemia and hyperglycemia in type 2 diabetes. In this study, we evaluated whether activation of PPARα and PPARγ stimulates neoangiogenesis. RESEARCH DESIGN AND METHODS—We used selective synthetic PPARα and PPARγ agonists and investigated their angiogenic potentials in vitro and in vivo. RESULTS—Activation of PPARα and PPARγ leads to endothelial tube formation in an endothelial/interstitial cell co-culture assay. This effect is associated with increased production of the angiogenic cytokine vascular endothelial growth factor (VEGF). Neovascularization also occurs in vivo, when PPARα and PPARγ agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPARα and PPARγ agonists are respectively used in PPARα knockout mice and mice treated with a specific PPARγ inhibitor, demonstrating that this angiogenic response is PPAR mediated. PPARα- and PPARγ-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPARα- and PPARγ-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPARα and PPARγ agonists, there is increased phosphorylation of endothelial nitric oxide synthase and Akt. CONCLUSIONS—These findings demonstrate that PPARα and PPARγ activation stimulates neoangiogenesis through a VEGF-dependent mechanism. Neoangiogenesis is a crucial pathological event in type 2 diabetes. The ability of PPARα and PPARγ agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of type 2 diabetes.

Sonic Hedgehog Regulates Angiogenesis and Myogenesis During Post-Natal Skeletal Muscle Regeneration

Sonic hedgehog (Shh) is a morphogen‐regulating crucial epithelial‐mesenchymal interactions during embryonic development, but its signalling pathway is considered generally silent in post‐natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant up‐regulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signalling pathway has an important regulatory role on injury‐induced angiogenesis, as inhibition of Shh function results in impaired up‐regulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal‐derived factor (SDF)‐1alpha, decreased muscle blood flow and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf‐5 and MyoD, decreases the up‐regulation of insulin‐like growth factor (IGF)‐1 and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post‐natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.

A general method for permeabilizing monolayer and suspension cultured animal cells.

Abstract A wide variety of animal cells have been successfully permeabilized to non-penetrating molecules, using lysolecithin. The sizes of molecules that can enter the cells can be controlled by varying the concentration of lysolecithin. The cells become permeable to small molecules and maintain viability following treatment with low lysolecithin concentrations. At higher concentrations the cells become permeable to proteins but do not retain viability. Lysolecithin permeabilization should permit many studies of the effects of non-penetrating compounds on cellular processes.

CCN5 Is a Growth Arrest-Specific Gene That Regulates Smooth Muscle Cell Proliferation and Motility

Vascular smooth muscle cell (VSMC) hyperplasia plays an important role in both chronic and acute vascular pathologies. Considerable work has focused on the mechanisms regulating VSMC growth and the search for agents that could suppress VSMC hyperproliferation. One of the several inhibitors studied is the glycosaminoglycan heparin, which inhibits VSMC proliferation and migration both in cell culture and in animal models (Mishra-Gorur K, Delmolino LM, Castellot Jr JJ: Biological functions of heparan sulfate and heparan sulfate proteoglycans. Trends Glycosci Glycotechnol 1998, 10:193-210). To aid our understanding of the anti-proliferative mechanism of action of heparin, we used a subtractive hybridization approach to isolate and characterize a novel growth arrest-specific (gas) gene induced in VSMCs exposed to heparin (Delmolino LM, Stearns NA, Castellot Jr JJ: Heparin induces a member of the CCN family which has characteristics of a growth arrest specific gene. Mol Biol Cell 1997, 8:287a and Delmolino LM, Stearns NA, Castellot Jr JJ: COP-1, a member of the CCN family, is a heparin-induced growth arrest specific gene in vascular smooth muscle cells. J Cell Physiol 2001, 188:45-55). This gene is a member of the cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed (CCN) family and has been given the name CCN5. In this report, we provide functional evidence that CCN5 can inhibit VSMC proliferation, motility, and invasiveness. In contrast, adhesion and apoptosis are unaffected by CCN5 in this cell type. We also significantly extend previous data from our laboratory that suggests CCN5 is a growth arrest-specific (gas) gene. Furthermore, we map for the first time the cellular localization of CCN5 protein in cultured VSMCs. We also examine uninjured and balloon-injured rat carotid arteries for CCN5 expression. The results from the in vitro and in vivo localization studies show that CCN5 is temporally and spatially expressed in a manner consistent with a role in regulating proliferation, motility, and invasiveness of VSMCs.

Pro‐inflammatory genetic profiles in subjects with peripheral arterial occlusive disease and critical limb ischemia

Objectives.  Single nucleotide polymorphisms in genes encoding inflammatory molecules may determine genetic profiles associated with increased risk of development and progression of cardiovascular diseases. In this study, we evaluated distribution and reciprocal interaction of a set of functionally important polymorphisms of genes encoding prototypical inflammatory molecules in subjects with peripheral arterial occlusive disease (PAOD) and critical limb ischemia (CLI). We also investigated whether synergistic interactions between these pro‐inflammatory gene polymorphisms influence the risk of PAOD and CLI.

COP-1, a member of the CCN family, is a heparin-induced growth arrest specific gene in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) hyperplasia is responsible for the failure of 15–30% of vascular surgical procedures such as coronary artery bypass grafts and angioplasties. We and others have shown that heparin suppresses VSMC proliferation in vivo and in cell culture. We hypothesize that heparin inhibits VSMC proliferation by binding to cell surface receptors, resulting in selective modulation of mitogenic signal transduction pathways and altered transcription of a specific subset of growth regulatory genes. To test this idea, we used subtractive hybridization to identify differentially expressed mRNAs in heparin‐treated and untreated VSMC. We identified a heparin induced mRNA identical to Cop‐1, a member of the CCN family of proteins which are secreted, cysteine‐rich modular proteins involved in growth regulation and migration. Cop‐1 from smooth muscle cells appears to have a different expression pattern and possibly different functions than Cop‐1 from other cells. Cop‐1 mRNA is expressed at high levels in quiescent VSMC and at low levels in proliferating VSMC, an expression pattern highly characteristic of growth arrest specific genes. Cop‐1 mRNA is expressed at high levels in heparin treated VSMC and COP‐1 protein is secreted into culture medium. In tissues, Cop‐1 expression is observed in the uninjured rat aorta suggesting a possible role for Cop‐1 in vivo. We found PDGF, but not EGF, inhibits the expression of Cop‐1 in VSMC. Neither TGF‐β nor interferon‐β, two inhibitors of VSMC proliferation, were able to induce Cop‐1 expression. In addition, heparin does not induce Cop‐1 mRNA in endothelial cells and VSMC resistant to the antiproliferative effect of heparin. Conditioned medium from cells over‐expressing COP‐1 protein inhibits VSMC proliferation in culture. Together, our data indicate that COP‐1 may play a role in the antiproliferative mechanism of action of heparin.

Heparin suppresses sgk, an early response gene in proliferating vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) hyperplasia plays a central role in chronic and acute vascular pathology including arteriosclerosis and restenosis following vascular surgery. The glycosaminoglycans of the heparan sulfate class, including heparin, inhibit VSMC proliferation in animals and in culture. Heparin binds to high affinity sites on the cell surface, selectively modulates mitogenic signal transduction pathway(s), and rapidly alters transcription of several genes. To further explore the molecular mechanisms responsible for this growth inhibition, we have employed the differential display technique to identify heparin‐regulated genes. Here we demonstrate that heparin inhibits the expression of the early response gene sgk (serum and glucocorticoid‐regulated kinase). The expression of sgk is not inhibited by chondroitin sulfate, a nonantiproliferative glycosaminoglycan, suggesting that sgk suppression may play a functional role in the antiproliferative effect of heparin. This idea is strengthened by the finding that heparin does not inhibit sgk expression in VSMCs resistant to the antiproliferative effect of heparin or in vascular endothelial cells which are unresponsive to heparin. Expression of sgk mRNA diminishes with increasing concentrations of heparin. Finally, sgk expression is not suppressed by other growth inhibitors such as transforming growth factor‐β1 (TGF‐β1) and interferon‐β (IFN‐β), suggesting separate and distinct effects of these growth inhibitors on the mitogenic pathway. J. Cell. Physiol. 173:371–379, 1997.