Alejandro Adam

To delineate signals by which the vascular abnormalities inherent to ocular rosacea arise and to correlate these signals with elements of the innate immune system. Experimental study. Immunohistochemical staining was performed for a variety of vascular markers and for toll-like receptor-4 on eyelid biopsies taken from patients with ocular rosacea and normal controls. Statistical comparisons were then performed between the 2 groups. Immunohistochemical staining for CD31 and integrin-β-3 did not demonstrate any statistically significant differences between eyelids from patients with ocular rosacea and normal controls. Cutaneous biopsies from ocular rosacea patients demonstrated statistically significant enrichments of intercellular adhesion molecule-1 and CD105 among arterioles, whereas there were no statistically significant differences in the venules between normal controls and ocular rosacea patients. The correlation between the number of toll-like receptor-4-positive cells and each vascular marker was statistically significant. Cutaneous biopsies of the eyelid did not demonstrate an increase in the total number of blood vessels. However, the vascular abnormalities that are typical of ocular rosacea represent activated, inflamed vessels, and these phenomena may be mediated by intercellular adhesion molecule and CD105. Furthermore, the strong correlations between toll-like receptor-4 and each vascular marker suggest that the innate immune system may govern the cutaneous effects of ocular rosacea. Intercellular adhesion molecule, CD105, and toll-like receptor-4 may represent important therapeutic targets in the management of this disease.

To characterize the molecular biologic environment of pathological skin in ocular rosacea and to differentiate the levels of inflammatory molecules in ocular rosacea from those of normal skin. The concentrations of 48 molecules were assayed in cutaneous biopsies taken from patients with ocular rosacea and from normal controls. There were very few molecular differences between the 2 groups, and 43 of the 48 molecules that were measured in this study were not significantly different between the 2 groups. The concentrations of 5 molecules (interleukin-1β, interleukin-16, stem cell factor, monocyte chemotactic protein-1, and monokine induced by γ-interferon) were significantly enriched in ocular rosacea. Ocular rosacea is a highly ordered molecular process and involves elevations in the concentrations of specific molecules. The particular pattern of enrichment supports the notion that ocular rosacea represents a disorder of innate immunity. Furthermore, these molecules may represent novel therapeutic targets in the future management of this disorder.

Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced ...

Leukocyte transendothelial migration (TEM) is regulated by several signaling pathways including Src family kinases (SFK) and the small RhoGTPases. Previous studies have shown that vascular endothelial-cadherin (VE-cad) forms a complex with β-,γ-, and p120-catenins and this complex disassociates to form a transient gap during leukocyte TEM. Additionally, p120-catenin (p120-1A) overexpression in human umbilical vein endothelial cells (HUVEC) stabilizes VE-cad surface expression, prevents tyrosine phosphorylation of VE-cad, and inhibits leukocyte TEM. Based on reports showing that p120 overexpression in fibroblasts or epithelial cells inhibits RhoA and activates Rac and Cdc42 GTPases, and on other reports showing that RhoA activation in endothelial cells is necessary for leukocyte TEM, we reasoned that p120 overexpression inhibited TEM through inhibition of RhoA. To test this idea, we overexpressed a mutant p120 isoform, p120-4A, which does not interact with RhoA. p120-4A colocalized with VE-cad in HUVEC junctions and enhanced VE-cad surface expression, similar to overexpression of p120-1A. Interestingly, overexpression of either p120-4A or p120-1A dramatically blocked TEM, and overexpression of p120-1A in HUVEC did not affect RhoA basal activity or activation of RhoA and Rac induced by thrombin or ICAM-1 crosslinking. In contrast, biochemical studies revealed that overexpression of p120-1A reduced activated pY416-Src association with VE-cad. In summary, p120 overexpression inhibits neutrophil TEM independently of an effect on RhoA or Rac and instead blocks TEM by preventing VE-cad tyrosine phosphorylation and association of active Src with the VE-cad complex.