Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

Endosialin Promotes Atherosclerosis Through Phenotypic Remodeling of Vascular Smooth Muscle Cells.

OBJECTIVE: Vascular smooth muscle cells (VSMC) play a key role in the pathogenesis of atherosclerosis, the globally leading cause of death. The transmembrane orphan receptor endosialin (CD248) has been characterized as an activation marker of cells of the mesenchymal lineage including tumor-associated pericytes, stromal myofibroblasts, and activated VSMC. We, therefore, hypothesized that VSMC-expressed endosialin may display functional involvement in the pathogenesis of atherosclerosis. APPROACH AND RESULTS: Expression of endosialin was upregulated during atherosclerosis in apolipoprotein E (ApoE)-null mice and human atherosclerotic samples analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry. Atherosclerosis, assessed by Oil Red O staining of the descending aorta, was significantly reduced in ApoE/endosialin-deficient mice on Western-type diet. Marker analysis of VSMC in lesions induced by shear stress-modifying cast implantation around the right carotid artery identified a more pronounced contractile VSMC phenotype in the absence of endosialin. Moreover, in addition to contributing to neointima formation, endosialin also potentially regulated the proinflammatory phenotype of VSMC as evidenced in surrogate cornea pocket assay experiments in vivo and corresponding flow cytometry and ELISA analyses in vitro. CONCLUSIONS: The experiments identify endosialin as a potential regulator of phenotypic remodeling of VSMC contributing to atherosclerosis. The association of endosialin with atherosclerosis and its absent expression in nonatherosclerotic samples warrant further consideration of endosialin as a therapeutic target and biomarker.

Hematoprotective effect of seleno-L-methionine on cyclophosphamide toxicity in rats.

Cyclophosphamide (CP) is a widely used antineoplastic drug that causes toxicity in the normal cell due to its metabolites. The major drawback of this drug is an undesirable myelosuppression. Selenium (Se) is a potent nutritional antioxidant that carries out biological effects by its incorporation into selenoproteins, such as glutathione peroxidase (GPx). The possible protective effects of seleno-L-methionine (SLM) against CP-related toxicity of blood cells and bone marrow of rats were investigated in this study. Intraperitoneal (i.p) administration of 50, 100, or 150 mg/kg of CP caused, in a dose-dependent manner, reductions in the number of leukocytes (78, 89, and 92%, respectively), thrombocytes (22, 33, and 52%, respectively), and bone marrow-nucleated cells (72, 90, and 94%, respectively). The groups that had CP treatment alone were killed 3 days after the CP injection. For the groups having CP+SLM, SLM (0.4 or 0.8 mg/kg i.p) administration was started 3 days earlier than the CP administration and continued to the end of the experiment (6 days). On day 4, the animals were weighed again, relative doses of CP were estimated, and CP+SLM was administered together. On day 7, blood samples were collected and bone marrow of animals were resected under anesthesia. The results indicated that treatment of rats within a select dose range of SLM could reduce CP-induced toxicity on blood cells and bone marrow.

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling.

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

Seleno L-methionine acts on cyclophosphamide-induced kidney toxicity.

The anticancer drug cyclophosphamide (CP) has nephrotoxic effects besides its urotoxicity, which both in turn limit its clinical utility. The nephrotoxicity of CP is less common compared to its urotoxicity, and not much importance has been given for the study of mechanism of CP-induced nephrotoxicity so far. Overproduction of reactive oxygen species (ROS) during inflammation is one of the reasons of the kidney injury. Selenoproteins play crucial roles in regulating ROS and redox status in nearly all tissues; therefore, in this study, the nephrotoxicity of CP and the possible protective effects of seleno L-methionine (SLM) on rat kidneys were investigated. Forty-two Sprague-Dawley rats were equally divided into six groups of seven rats each. The control group received saline, and other rats were injected with CP (100 mg/kg), SLM (0.5 or 1 mg/kg), or CP + SLM intraperitoneally. Malondialdehyde (MDA) and glutathione (GSH) levels in kidney homogenates of rats were measured, and kidney tissues were examined under the microscope. CP-treated rats showed a depletion of renal GSH levels (28% of control), while CP + SLM-injected rats had GSH values close to the control group. MDA levels increased 36% of control following CP administration, which were significantly decreased after SLM treatment. Furthermore, these biochemical results were supported by microscopical observations. In conclusion, the present study not only points to the therapeutic potential of SLM in CP-induced kidney toxicity but also indicates a significant role for ROS and their relation to kidney dysfunction.

Protective effect of seleno-L-methionine on cyclophosphamide-induced urinary bladder toxicity in rats.

Cyclophosphamide (CP) is a widely used antineoplastic drug, which could cause toxicity of the normal cells due to its toxic metabolites. Its urotoxicity may cause dose-limiting side effects like hemorrhagic cystitis. Overproduction of reactive oxygen species (ROS) during inflammation is one of the reasons of the urothelial injury. Selenoproteins play crucial roles in regulating ROS and redox status in nearly all tissues; therefore, in this study, the urotoxicity of CP and the possible protective effects of seleno-L: -methionine (SLM) on urinary bladder of rats were investigated. Intraperitoneal (i.p.) administration of 50, 100, or 150 mg/kg CP induced cystitis, in a dose-dependent manner, as manifested by marked congestion, edema and extravasation in rat urinary bladder, a marked desquamative damage to the urothelium, severe inflammation in the lamina propria, focal erosions, and polymorphonuclear (PMN) leukocytes associated with occasional lymphocyte infiltration determined by macroscopic and histopathological examination. In rat urinary bladder tissue, a significant decrease in the endogenous antioxidant compound glutathione, and elevation of lipid peroxidation were also noted. Pretreatment with SLM (0.5 or 1 mg/kg) produced a significant decrease in the bladder edema and caused a marked decrease in vascular congestion and hemorrhage and a profound improvement in the histological structure. Moreover, SLM pretreatment decreased lipid peroxide significantly in urinary bladder tissue, and glutathione content was greatly restored. These results suggest that SLM offers protective effects against CP-induced urinary bladder toxicity and could be used as a protective agent against the drug toxicity.

Protective effect of zinc on cyclophosphamide-induced hematoxicity and urotoxicity.

Cyclophosphamide (CP) is widely used for the treatment of neoplastic diseases; however, its toxicity causes dose-limiting side effects. Zinc (Zn) is an essential trace element and has important biological functions that control many cell processes including DNA synthesis, normal growth, reproduction, fetal development, bone formation, and wound healing. Therefore, the toxicity of CP and the possible protective effect of Zn on blood cells, bone marrow, and bladder of rat were investigated in this study. Intraperitoneal administration of 50, 100, or 150 mg/kg CP for 3 days caused, in a dose-dependent manner, reductions in the number of leukocytes, thrombocytes, and bone marrow nucleated cells and a serious urotoxicity. To explore whether CP-induced damages could be prevented by Zn, other groups of rats were pretreated with 4 or 8 mg/kg ZnCl2 intraperitoneally for 3 days then challenged with respective doses of CP plus ZnCl2 on day 4 for three more days. The results indicated that treatment of rats with Zn could dose-dependently alleviate CP-induced toxicities on blood cells, bone marrow cells, and urinary bladder. We suggest that Zn could be a potentially effective drug in the prevention of CP-related hematoxicity and urotoxicity.