Angiogenesis is an important process in maintaining normal physiology as well as in the pathology of many diseases. Angiogenesis based therapies have the potential to have a phenomenal impact on a diseases affecting more than one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, complications of AIDS, diabetes, Alzheimer's disease, and more than 70 other major health conditions affecting children and adults, in developed and developing nations. In this study, we investigate the role of Interleukin-19 (IL-19) and Allograft inflammatory factor-1 (AIF-1) in endothelial cells (EC) proliferation, migration, activation and angiogenic potential. IL-19 is a recently described member of the IL-10 family of anti-inflammatory cytokines. Nothing has been reported on the expression or mechanism(s) of IL-19 effects in endothelial cells. We have found that IL-19 is expressed in aortic endothelium, and can be induced in cultured EC by serum and inflammatory cytokine challenge. IL-19 is chemotactic for EC, and promotes cell spreading, migration, and wound healing. IL-19 pretreatment also enhances the migration of EC to Vascular endothelial growth factor (VEGF). IL-19 activates the signaling proteins STAT3, p44/42 MAPK, and Rac1, and induces expression of Matrix Metalloproteinase-2, which is obligate for EC migration. IL-19 promotes tube -like structure formation on Matrigel by Human Umbilical Vein EC (HUVEC), and promotes microvessel formation in mouse aortic ring assay. Taken together, these data suggest that IL-19 is a chemotactic cytokine for, and promotes the proliferation, migration, activation and angiogenic potential of endothelial cells. AIF-1 is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that has been implicated in the regulation of inflammation. The expression and function of AIF-1 in EC is uncharacterized. AIF-1 expression co-localized with CD31-positive endothelial cells in neointima of inflamed human arteries, but not normal arteries. AIF-1 is detected at low levels in unstimulated EC, but expression can be increased in response to serum and soluble factors. Stable transfection of AIF-1 siRNA in EC reduced AIF-1 protein expression by 73%, and significantly reduced EC proliferation and migration. Rescue of AIF-1 expression restored both proliferation and migration of siRNA expressing ECs, and AIF-1 over expression enhanced both of these activities, suggesting a strong association between AIF-1 expression and EC activation. Activation of MAPK p44/42 and PAK1 was significantly reduced in siRNA ECs challenged with inflammatory stimuli. Reduction of AIF-1 expression did not decrease EC tube-like structure or microvessel formation from aortic rings, but over-expression of AIF-1 did significantly increase the number and complexity of these structures. These data indicate that AIF-1 expression plays an important role in proliferation, migration, signal transduction, and may have a role in angiogenesis. / Microbiology and Immunology

The underlying cause of all vascular proliferative diseases is injury-induced activation of vascular endothelium and vascular smooth muscle cells (VSMC). Activated VSMC proliferate, than migrate from the arterial media to the intima, contributing to neointima formation. Activated immune cells, vascular cells, and their endogenous regulators mediate this complex process. One integral regulator of VSMC activation is allograft inflammatory factor-1 (AIF-1). AIF-1 is a cytoplasmic scaffold protein, expressed constitutively in lymphoid cells and induced in VSMC by injury. Stable over expression of AIF-1 increases VSMC proliferation and migration in vitro, causes increased injury-induced neointima formation, and increases Rac1 and p38 MAP Kinase activity. Recent studies show a correlation between VSMC expression of AIF-1 and atherosclerosis development. We hypothesize that VSMC over expression of AIF-1 contributes to atherosclerosis development by increasing activity of inflammatory signaling molecules, and that inhibiting VSMC AIF-1 expression will decrease injury-induced neointima formation. Rat carotid arteries transfected with AIF-1 si RNA adenovirus after balloon angioplasty developed significantly less neointima compared to controls. AIF-1 si RNA transfected VSMC proliferated significantly less than AIF-1 or GFP transfected VSMC, while AIF-1 si RNA transfection did not attenuate AIF-1-mediated migration. p38 inhibition showed that AIF-1-mediated proliferation is dependent on p38 activation while AIF-1-mediated migration is not. AIF-1 transgenic mice fed a high fat diet showed significantly more atherosclerotic lesions than WT littermates. Boyden Chamber assays showed OxLDL treatment increases VSMC migration but does not effect AIF-1-mediated migration. Expression of migration and inflammatory responsive genes in AIF-1 and XGal transfected VSMC after OxLDL treatment at various time points were examined. MMP-2 and -9 expression did not change. ICAM-1 and VCAM-1 expression increased in both groups. AIF-1 VSMC showed significantly higher ICAM-1 expression at baseline and early time points and elevated, but not significantly higher VCAM-1 expression at early time points. Western blots showed increased activation of NF-kB in AIF-1 transfected VSMC at baseline and 30 minutes after OxLDL stimulation compared to XGal transfected VSMC. Expression of the scavenger receptor receptors CD36 and SRA(I) expression increased after lipid treatment in AIF-1 and XGal transfected groups. AIF-1 VSMC showed sustained expression of both receptors after 16 hours of treatment compared to XGal VSMC, which showed decreased expression at that time point. CXCL16/PSOX expression increased with treatment, but differences in expression patterns were not seen between cell groups. Analysis showed significantly more OxLDL was taken up by AIF-1 VSMC compared to XGal VSMC. These data show that AIF-1 expression in VSMC is tightly linked to the vascular response to injury and development of vascular disease. Although AIF-1-mediated migration is not p38 dependent, AIF-1 may contribute to increased VSMC migration in part by upregulating NF- kB downstream effectors through increased NF-kB activity. AIF-1 may also speed the progression of atherosclerosis by increasing scavenger receptor expression and thereby increasing OxLDL uptake and foam cell formation. Although more study is required to fully elucidate the molecular mechanisms leading to AIF-1 mediated VSMC activation, these data have further established AIF-1 as an integral regulator of the VSMC response to injury. / Molecular and Cellular Physiology