Endothelium-derived microparticles have recently been described as a new marker of endothelial cell dysfunction. Increased levels of circulating microparticles have been documented in inflammatory disorders, diabetes mellitus, and many cardiovascular diseases. Perturbations of angiogenesis play an important role in the pathogenesis of these disorders. We demonstrated previously that isolated endothelial microparticles (EMPs) impair endothelial function in vitro, diminishing acetylcholine-induced vasorelaxation and nitric oxide production by rat aortic rings and simultaneously increasing superoxide production. Herein, using the Matrigel assay of angiogenesis in vitro and a topological analysis of the capillary-like network by human umbilical vein endothelial cells (HUVECs), we investigated the effects of EMPs on formation of the vascular network. All parameters of angiogenesis were affected by treatment for 48 h with isolated EMPs in a concentration of 10(5) but not 10(3) or 10(4) EMPs/ml. The effects included decreases in total capillary length (24%), number of meshes (45%), and branching points (36%) and an increase in mesh area (38%). The positional and topological order indicated that EMPs affect angiogenic parameters uniformly over the capillary network. Treatment with the cell-permeable SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (Mn-TBAP) partially or completely restored all parameters of angiogenesis affected by EMPs. EMPs reduced cell proliferation rate and increased apoptosis rate in time- and dose-dependent manners, and this phenomenon was also prevented by Mn-TBAP treatment. Our data demonstrate that EMPs have considerable impact on angiogenesis in vitro and may be an important contributor to the pathogenesis of diseases that are accompanied by impaired angiogenesis.

Heme oxygenase isoforms (HO-1/HO-2) catalyze the conversion of heme to carbon monoxide (CO) and bilirubin. In this study, HO-1-deficient endothelial cells were transduced with HO-1 in the antisense orientation to determine whether supplementation with CO or bilirubin would regulate cell proliferation and angiogenesis. Western blotting, enzyme activity, CO and prostaglandin E(2) (PGE(2)) production, and cell-cycle analysis were used to assess transgenic expression and functionality of the recombinant protein. A Matrigel matrix was used for assessment of in vitro capillary formation. Transduction with HO-1 antisense resulted in decreased capillary formation, cell proliferation, and cell-cycle progression, and increased PGE(2) production compared with control. HO-1 deficiency was also associated with increased expression of p21 and p27, but had no significant effect on p16 and p53. We also compared two different CO donors for their ability to rescue angiogenesis. Compared with control, HO-1-deficient endothelial cells showed increased angiogenesis following tricarbonyldichlororuthenium( II) dimer ([Ru(CO)(3)Cl(2)](2)) (CORM-1) starting at 50 microM, whereas tricarbonylchloro(glycinato) ruthenium(II) (CORM-3), starting at 25 microM, was a potent enhancer of angiogenesis. The addition of bilirubin did not restore angiogenesis. These data suggest that HO-mediated angiogenesis and cell proliferation were dependent on HO-1- and not HO-2-derived CO.

The molecular mechanisms of skin adaptation to the environmental stress are poorly understood. The aryl hydrocarbon receptor nuclear translocator (Arnt) lies at the intersection of several crucial adaptive pathways. Nevertheless, its role in adaptation of the skin to environmental stress has just begun to be unraveled. Here we show that Arnt is expressed in human and mouse skin in a developmentally dependent manner. Targeted K14-driven deletion of Arnt in the mouse epidermis resulted in early postnatal death, associated with a failure of epidermal barrier function. Gene expression profiling of Arnt-null mouse epidermis revealed upregulation of genes of the epidermal differentiation complex on mouse chromosome 3, including S100a genes (S100a8, S100a9, S100a10) and genes coding for small proline-rich proteins (Sprr1a, Sprr2i, Sprr2j, Sprrl1). HPTLC analysis showed significant accumulation of Cer[NS] and Cer[NH] ceramide species in Arnt-null epidermis, suggesting alterations in lipid metabolism. Continuous retention of corneosomes in Arnt-null epidermis that resulted in an abnormally dense corny layer and impaired desquamation was associated with upregulation of Slpi, an inhibitor of stratum corneum chymotryptic enzyme (SCCE) that plays a key role in corneosome degradation. The functional defects in Arnt-null mouse epidermis underscore the crucial role of Arnt in the maintenance of epidermal homeostasis, especially during the perinatal transition to the ex utero environment.

Accumulating data suggest that the biological responses to high and low doses of radiation are qualitatively different, necessitating the direct study of low-dose responses to better understand potential risks. Most such studies have used two-dimensional culture systems, which may not fully represent responses in three-dimensional tissues. To gain insight into low-dose responses in tissue, we have profiled global gene expression in EPI-200, a three-dimensional tissue model that imitates the structure and function of human epidermis, at 4, 16 and 24 h after exposure to high (2.5 Gy) and low (0.1 Gy) doses of low-LET protons. The most significant gene ontology groups among genes altered in expression were consistent with effects observed at the tissue level, where the low dose was associated with recovery and tissue repair, while the high dose resulted in loss of structural integrity and terminal differentiation. Network analysis of the significantly responding genes suggested that TP53 dominated the response to 2.5 Gy, while HNF4A, a novel transcription factor not previously associated with radiation response, was most prominent in the low-dose response. HNF4A protein levels and phosphorylation were found to increase in tissues and cells after low- but not high-dose irradiation.

Abstract-Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid -hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell-specific promoter SM22␣ (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). ␤-Galactosidase expression was detected in Ad-SM22-nLacZ-transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1-transduced A7r5 and PAC1 cells. Ad-SM22-4A1-transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79Ϯ1.77 versus 0.97Ϯ0.57 nmol 12-hydroxy lauric acid/10 6 cells per h). Smooth muscle-specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04Ϯ0.29 and 13.43Ϯ2.84 ng/mg protein in Ad-SM22-nLacZ-transduced and Ad-SM22-4A1-transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63Ϯ3.66 mm in Ad-SM22-4A1-transduced vessels versus 1.79Ϯ0.89 mm in Ad-SM22-nLacZ-transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle-specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.