Reduction of Adipose Tissue Mass by the Angiogenesis Inhibitor ALS-L1023 from Melissa officinalis.

It has been suggested that angiogenesis modulates adipogenesis and obesity. This study was undertaken to determine whether ALS-L1023 (ALS) prepared by a two-step organic solvent fractionation from Melissa leaves, which exhibits antiangiogenic activity, can regulate adipose tissue growth. The effects of ALS on angiogenesis and extracellular matrix remodeling were measured using in vitro assays. The effects of ALS on adipose tissue growth were investigated in high fat diet-induced obese mice. ALS inhibited VEGF- and bFGF-induced endothelial cell proliferation and suppressed matrix metalloproteinase (MMP) activity in vitro. Compared to obese control mice, administration of ALS to obese mice reduced body weight gain, adipose tissue mass and adipocyte size without affecting appetite. ALS treatment decreased blood vessel density and MMP activity in adipose tissues. ALS reduced the mRNA levels of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9), whereas ALS increased the mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1, and TIMP-2) in adipose tissues. The protein levels of VEGF, MMP-2 and MMP-9 were also decreased by ALS in adipose tissue. Metabolic changes in plasma lipids, liver triglycerides, and hepatic expression of fatty acid oxidation genes occurred during ALS-induced weight loss. These results suggest that ALS, which has antiangiogenic and MMP inhibitory activities, reduces adipose tissue mass in nutritionally obese mice, demonstrating that adipose tissue growth can be regulated by angiogenesis inhibitors.

ALH-L1005 attenuates endotoxin induced inner ear damage.

OBJECTIVE: To assess whether this compound (ALH-L1005) is conceivably an effective agent in protecting against cochlear damage induced by LPS. MATERIALS AND METHODS: Tube formation using human umbilical vein endothelial cell (HUVEC) and matrix metalloproteinase (MMP)-9 inhibition assay was performed. 24 guinea pigs were randomly divided into three groups. Intratympanic instillation of LPS (n=8) as negative control, instillation of oxytetracycline 1h after LPS as positive control (n=8), and intratympanic instillation of ALH-L1005 (n=8) 1h after LPS were considered experimental group. Evaluation by auditory brainstem response (ABR) measurement, cochlear blood flow, and blood-labyrinth barrier (BLB) permeability were performed. Cochlear hair cells were observed by field emission-scanning electron microscopy (FE-SEM). MMP-9 activation was measured by gelatin zymography. RESULTS: For HUVEC, the tube formation was suppressed in a dose dependant manner. ALH-L1005 inhibited the MMP-9 activity prominently. It also attenuated the elevation of LPS-induced hearing threshold shift and recovery of CBF. By FE-SEM, cochlear hair cells could be preserved in experimental group. ALH-L1005 significantly reduced the BLB opening compared to LPS group. Active MMP-9 expression could be detected in the LPS group. In contrast to ALH-L1005 group, active MMP-9 expression was not detected. CONCLUSION: Our results conclude that ALH-L1005 showed a protective effect in the cochlear lateral wall damage induced by LPS.

Matrix metalloproteinase inhibitor attenuates cochlear lateral wall damage induced by intratympanic instillation of endotoxin.

OBJECTIVE: Oxytetracycline and ilomastat are inhibitors of matrix metalloproteinases (MMPs). Their efficacy in protecting against cochlear damage induced by the intratympanic instillation of lipopolysaccharide (LPS), as a means of inducing labyrinthitis, was investigated. MATERIALS AND METHODS: Experiments were performed in 21 young male guinea pigs. Intratympanic instillation of LPS was done in the control group (n=7). Intratympanic instillation of oxytetracycline or ilomastat was done after LPS instillation in the experimental group. Measurements of auditory brainstem response (ABR) and cochlear blood flow (CBF) were performed. The organ of Corti was evaluated by field emission scanning electron microscopy (FE-SEM). The blood-labyrinth barrier (BLB) integrity was evaluated with Evans blue uptake. Gelatin zymography was used to assess the expression of active MMP-2 and MMP-9. RESULTS: Ears treated with MMP inhibitors were significantly protected from hearing loss compared to the LPS group. In LPS group, there was a significant decrease of CBF. However, experimental group displayed a statistically significant recovery of CBF. FE-SEM revealed hair cell damage in the LPS-treated group, but hair cells presented a normal appearance in MMP inhibitors. The LPS group showed a marked increase of Evans blue extravasation in the cochlea. However, MMP inhibitors significantly reduced the BLB opening. Active MMP-9 was expressed in the LPS group. Treatment with MMP inhibitors attenuated active MMP-9 expression. CONCLUSION: The MMP inhibitors oxytetracycline and ilomastat protect from cochlear lateral wall damage caused by LPS-induced labyrinthitis.

Regulation of obesity and lipid disorders by herbal extracts from Morus alba, Melissa officinalis, and Artemisia capillaris in high-fat diet-induced obese mice.

Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae) are suggested to be involved in the regulation of hyperlipidemia. We hypothesized that Ob-X, a mixture of three herbs, Morus alba, Melissa officinalis and Artemisia capillaris [corrected] improves lipid metabolism, body weight gain and adiposity and that peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with these events. Mice fed a high-fat diet for 12 weeks exhibited increases in body weight gain and adipose tissue mass compared with mice fed a low fat diet. However, feeding a high-fat diet supplemented with Ob-X significantly reduced these effects. Ob-X treatment also decreased the circulating levels of triglycerides and total cholesterol, and inhibited hepatic lipid accumulation. Ob-X supplementation was found to increase the hepatic mRNA levels of PPARalpha target enzymes responsible for fatty acid beta-oxidation. Moreover, Ob-X elevated the endogenous expression of a luciferase reporter gene containing three copies of a PPAR response element (PPRE) in NMu2Li liver cells. These data demonstrate that Ob-X regulates body weight gain, adipose tissue mass, and lipid metabolism in part through changes in the expression of hepatic PPARalpha target genes.

A biologically active angiogenesis inhibitor, human serum albumin-TIMP-2 fusion protein, secreted from Saccharomyces cerevisiae.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous and bi-functional inhibitor of angiogenesis. TIMP-2 is expressed in an insoluble form in Escherichia coli and secreted at a very low level from yeast. Here, we report on a high level of secretion of TIMP-2 fused with human serum albumin (HSA) from the yeast Saccharomyces cerevisiae. The secreted HSA-TIMP-2 fusion protein (87kDa) was purified to greater than 95% homogeneity. The HSA-TIMP-2 protein inhibited approximately 81% of tube formation of human umbilical vein endothelial cells (HUVECs) when studied at a concentration of 187microM. The systemic administration of HSA-TIMP-2 at 40mg/kg to the C57B1/6 mouse inhibited the growth of B16BL6 tumors. Furthermore, a combination treatment of HSA-TIMP-2 with 5-fluorouracil (50mg/kg) showed significant effects on tumor growth in this model. The high level of secretion of the biologically active angiogenesis inhibitor from S. cerevisiae should facilitate fundamental research and application studies of HSA-TIMP-2, as an attractive candidate for therapeutic agents treating angiogenesis-related diseases.