mRNA Levels Of Angiogenic Factors Research Articles

α1-Adrenergic receptor mediates adipose-derived stem cell sheet-induced protection against chronic heart failure after myocardial infarction in rats

Cell-based therapy using adipose-derived stem cells (ADSCs) has emerged as a novel therapeutic approach to treat heart failure after myocardial infarction (MI). The purpose of this study was to determine whether inhibition of α1-adrenergic receptors (α1-ARs) in ADSCs attenuates ADSC sheet-induced improvements in cardiac functions and inhibition of remodeling after MI. ADSCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ADSCs, we determined the mRNA levels of vascular endothelial growth factor (VEGF)-A and α1-AR under normoxia or hypoxia and the effects of norepinephrine and an α1-blocker, doxazosin, on the mRNA levels of angiogenic factors. Hypoxia increased α1-AR and VEGF mRNA levels in ADSCs. Norepinephrine further increased VEGF mRNA expression under hypoxia; this effect was abolished by doxazosin. Tube formation of human umbilical vein endothelial cells was promoted by conditioned media of ADSCs treated with the α1 stimulant phenylephrine under hypoxia but not by those of ADSCs pretreated with phenylephrine plus doxazosin. In in vivo studies using rats with MI, transplanted ADSC sheets improved cardiac functions, facilitated neovascularization, and suppressed fibrosis after MI. These effects were abolished by doxazosin treatment. Pathway analysis from RNA sequencing data predicted significant upregulation of α1-AR mRNA expression in transplanted ADSC sheets and the involvement of α1-ARs in angiogenesis through VEGF. In conclusion, doxazosin abolished the beneficial effects of ADSC sheets on rat MI hearts as well as the enhancing effect of norepinephrine on VEGF expression in ADSCs, indicating that ADSC sheets promote angiogenesis and prevent cardiac dysfunction and remodeling after MI via their α1-ARs.

Human umbilical cord mesenchymal stem cells reconstruct the vaginal wall of ovariectomized Sprague-Dawley rats: implications for pelvic floor reconstruction.

Vaginal structural defects are involved in pelvic organ prolapse (POP). We tested whether mesenchymal stem cell (MSC) therapy can repair the weakened vaginal wall of POP patients as a novel POP treatment. Ninety-six ovariectomized rats were divided into 4 groups (n = 24/group): saline (sal), collagen (col), sal + MSC, and col + MSC groups. Two weeks after ovariectomy, rats received subepithelial injection of 0.3ml saline, 0.3ml collagen I gel, and 0.3ml saline: 3 × 106 human umbilical cord mesenchymal stem cells (HUMSCs), or 0.3ml collagen I gel: 3 × 106 HUMSCs into the anterior vaginal wall. Eight additional rats underwent in vivo bioluminescence imaging (BLI) to evaluate in vivo cell viability. The BLI signal disappeared within 1week after MSC injection, and no in vivo MSC differentiation was found. Collagen I content was significantly lower at 4 and 12weeks in the two MSC groups than in the sal and col groups, while collagen III was significantly higher (P < 0.001). The fraction of smooth muscle in the nonvascular muscularis increased significantly in the two MSC groups at 12weeks (P < 0.001). ACTA2 mRNA in the col + MSC group was significantly higher than that in the sal group at 2 and 4weeks (P = 0.042 and P = 0.040). mRNA levels of angiogenic factors (bFGF or VEGF) in the two MSC groups were significantly higher than those in the sal and col groups at different time points. HUMSCs normalized the fibromuscular structures of the vaginal wall of ovariectomized rats potentially through a paracrine effect.

597-P: Prolonged Therapeutic Effects of Photoactivated Adipose-Derived Stem Cells following Diabetic Ischemic Injury

Aim: Adipose-derived stem cells (ADSCs) therapies are emerging as a promising approach to therapeutic angiogenesis. Therapeutic persistence and reduced primitive stem cell function following cell delivery remains a critical hurdle for the clinical translation of stem cells in current approaches. Methods: Cultured ADSCs were derived from subcutaneous white adipose tissue isolated from mice fed a normal diet (ND). Photoactivated and non-light-treated ADSCs were injected into the tail vein of mice fed a high-fat diet (HFD), following which unilateral hindlimb ischemia was induced. Laser Doppler imaging was conducted to measure the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. mRNA levels of angiogenic factors were determined by reverse-transcription polymerase chain reaction, Flow cytometry was used to determine the characterization of ADSCs and endothelial progenitor cell (EPC). Human ADSCs secretomes were analyzed by liquid chromatography tandem mass spectrometry. Results: Our study demonstrated that photoactivated ND-ADSCs prolonged functional blood flow perfusion and increased ADSCs-derived EPC and neovascularization thirty-eight days after ligation, when compared with saline-treated controls. Profiling analysis in ischemic muscles showed upregulation of genes associated with pro-angiogenic factors after injection of photoactivated ND-ADSCs when compared with the non-light-treated ADSCs or saline treated HFD mice. Mass spectrometry revealed that light-treated ADSCs conditioned medium retained a more complete pro-angiogenic activity with significant upregulation of angiogenesis related proteins. Conclusion: Our data demonstrates that photoactivated ND-ADSCs improve blood flow recovery and their injection may prove to be a useful strategy for the prevention and treatment of diabetic peripheral arterial disease. Disclosure X. Fan: None. J. Wu: Research Support; Self; HarmonyRegena Inc.

Prolonged therapeutic effects of photoactivated adipose-derived stem cells following ischaemic injury.

Adipose-derived stem cells (ASCs) therapies are emerging as a promising approach to therapeutic angiogenesis. Therapeutic persistence and reduced primitive stem cell function following cell delivery remains a critical hurdle for the clinical translation of stem cells in current approaches. Cultured ASCs were derived from subcutaneous white adipose tissue isolated from mice fed a normal diet (ND). Unilateral hindlimb ischaemia model was induced in high-fat diet (HFD)-fed mice by femoral artery interruption, after which photoactivated and non-light-treated ASCs were injected into the tail vein of mice. Laser Doppler imaging was conducted to measure the blood flow reperfusion. Capillary density was measured in the ischaemic gastrocnemius muscle. mRNA levels of angiogenic factors were determined by reverse-transcription polymerase chain reaction. Flow cytometry was used to determine the characterization of ASCs and endothelial progenitor cell (EPC). Human ASCs secretomes were analysed by liquid chromatography tandem mass spectrometry. Our study demonstrated that photoactivated ND-ASCs prolonged functional blood flow perfusion and increased ASCs-derived EPC and neovascularization 38days after ligation, when compared with saline-treated controls. Profiling analysis in ischaemic muscles showed upregulation of genes associated with pro-angiogenic factors after injection of photoactivated ND-ASCs when compared with the non-light-treated ASCs or saline treated HFD mice. Mass spectrometry revealed that light-treated ASCs conditioned medium retained a more complete pro-angiogenic activity with significant upregulation of angiogenesis related proteins. Our data demonstrates that photoactivated ND-ASCs improve blood flow recovery and their injection may prove to be a useful strategy for the prevention and treatment of diabetic peripheral arterial disease.

β-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction.

Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.Methods and Results:ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a β-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts. ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

The balance between proliferation and transcription of angiogenic factors of mesenchymal stem cells in hypoxia

ABSTRACTBridging large bone defects with mesenchymal stromal cells-seeded scaffolds remains a big challenge in orthopedic surgery, due to the lack of vascularization. Within such a cell–scaffold construct, cells are exposed to ischemic conditions. When human mesenchymal stem cells (hMSCs) encounter hypoxic conditions, they show higher cell proliferation than at ambient oxygen levels. However, when hMSCs are exposed to prolonged ischemia, cell proliferation ceases completely. Exposure of hMSCs to hypoxic conditions is known to result in the transcription of angiogenic factors (AGF), which can promote the development of new blood vessels. In this study, we investigated at which oxygen level hMSC proliferation and the transcription of AGF were optimal. Human bone marrow-derived hMSCs were cultured at 0.1, 1, 2, 3, 4, 5, and 21% oxygen. Cell proliferation over 14 days was assayed using a DNA quantification method. hMSC metabolic activity over 14 days was measured using a MTT test. Quantitative RT-PCR was used to assess mRNA levels of angiogenic factors at the tested oxygen percentages. hMSCs showed the highest cell proliferation rate at 1% oxygen. The highest corrected cell metabolic rate was found at 21% oxygen, followed by 2% oxygen. HIF1α transcription did not increase under hypoxic conditions compared to 21% oxygen conditions. However, transcription of VEGF and ANG-1 was significantly higher at 2% oxygen than at 21% O2. The optimum oxygen range at which hMSCs proliferated rapidly and angiogenic factors ANG-1 and VEGF simultaneously came to expression was from 1 to 2% oxygen.

Involvement of NADPH oxidases in alkali burn-induced corneal injury

Chemical burns are a major cause of corneal injury. Oxidative stress, inflammatory responses and neovascularization after the chemical burn aggravate corneal damage, and lead to loss of vision. Although NADPH oxidases (Noxs) play a crucial role in the production of reactive oxygen species (ROS), the role of Noxs in chemical burn-induced corneal injury remains to be elucidated. In the present study, the transcription and expression of Noxs in corneas were examined by RT-qPCR, western blot analysis and immunofluorescence staining. It was found that alkali burns markedly upregulated the transcription and expression of Nox2 and Nox4 in human or mouse corneas. The inhibition of Noxs by diphenyleneiodonium (DPI) or apocynin (Apo) effectively attenuated alkali burn-induced ROS production and decreased 3-nitrotyrosine (3-NT) protein levels in the corneas. In addition, Noxs/CD11b double-immunofluorescence staining indicated that Nox2 and Nox4 were partially co-localized with CD11b. DPI or Apo prevented the infiltration of CD11b-positive inflammatory cells, and inhibited the transcription of inflammatory cytokines following alkali burn-induced corneal injury. In our mouse model of alkali burn-induced corneal injury, corneal neovascularization (CNV) occurred on day 3, and it affected 50% of the whole area of the cornea on day 7, and on day 14, CNV coverage of the cornea reached maximum levels. DPI or Apo effectively attenuated alkali burn-induced CNV and decreased the mRNA levels of angiogenic factors, including vascular endothelial growth factor (VEGF), VEGF receptors and matrix metalloproteinases (MMPs). Taken together, our data indicate that Noxs play a role in alkali burn-induced corneal injury by regulating oxidative stress, inflammatory responses and CNV, and we thus suggest that Noxs are a potential therapeutic target in the future treatment of chemical-induced corneal injury.

Effects of Korean red ginseng (Panax ginseng) on obesity and adipose inflammation in ovariectomized mice

Ethnopharmacological relevanceKorean red ginseng (ginseng, Panax ginseng C.A. Meyer) is a famous traditional drug used in Korea for the treatment and prevention of obesity, type 2 diabetes, cancer, and liver and cardiovascular diseases. Menopause is strongly associated with many of the aforementioned metabolic diseases and increased visceral obesity. The aims of this study were to investigate whether ginseng inhibits obesity and related disorders in ovariectomized (OVX) C57BL/6J mice, which is a mouse model of postmenopausal women, and to determine the mechanism of action involved in this process. Materials and methodsAfter OVX mice were treated with 5% (w/w) ginseng for 15 weeks, we determined the effects of ginseng on obesity and adipose inflammation, angiogenesis, metalloproteinase (MMP) activity and metabolic parameters. ResultsOVX mice had higher body weight, adipose tissue mass and adipocyte size when fed a high fat diet (HFD) compared with HFD-fed sham-operated mice. All of these parameters were significantly reduced in OVX mice fed a HFD supplemented with ginseng. Ginseng treatment also decreased blood vessel density, MMP activity, and mRNA levels of angiogenic factors (e.g., VEGF-A and FGF-2) and MMPs (e.g., MMP-2 and MMP-9) in adipose tissues of OVX mice. Infiltrating inflammatory cells and expression of inflammatory cytokines (e.g., CD68, TNFα and MCP-1) in adipose tissue were reduced by ginseng. Ginseng not only reduced the circulating levels of free fatty acids and triglycerides, but also normalized hyperinsulinemia and hyperglycemia in OVX mice. Hepatic lipid droplets were almost completely abolished by ginseng. ConclusionsThese results suggest that ginseng inhibited ovariectomy-induced obesity, adiposity, and adipocyte hypertrophy by modulating angiogenesis and MMP activity. Ginseng also suppressed adipose inflammation, insulin resistance, and hepatic steatosis in OVX mice. Thus, it is likely that ginseng may be a promising drug for the prevention and treatment of obesity and related disorders in obese postmenopausal women.

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.

Cardiac Adaptation To Exercise During Pregnancy

The extracellular composition and cardiac capillary density determine physiological from pathological cardiac hypertrophy. A number of studies demonstrate that exercise training does not increase fibrosis, whereas exercise training does increase capillary density and antioxidant capacity. However, whether exercise during pregnancy in previous sedentary dams could modify these parameters has not been investigated. METHODS AND RESULTS: 10 week old C57Bl/6 female mice were divided into non-pregnant diestrus control (C), late pregnancy (LP), and exercise during pregnancy (EX+LP). The EX+LP group was subjected to voluntary wheel running from gestation day 1 to 17. The pattern of running activity was high in early pregnancy and gradually decreased to low levels after mid-pregnancy. We found that both LP and EX+LP groups significantly increased heart mass normalized to tibial length (HW/TL). However, there were no differences in cardiac hypertrophy between LP and EX+LP. Next, we measured extracellular remodeling genes, angiogenic factors, and antioxidant related genes by quantitative real time PCR. mRNA levels of collagens, including Col1a1, Col3a1, and Col8a1, and connective tissue growth factor (Ctgf) were not altered in both LP and EX+LP groups compared to C. In addition, mRNA levels of angiogenic factors, including PGC-1α (Pgc1a), vascular endothelial growth factor a (Vegfa), angiogpietin-1 (Angpt1), Angpt2, fibroblast growth factor 2 (Fgf2), and platelet-derived growth factor alpha (Pdgfa) were not altered as well as antioxidant enzymes in both LP and EX+LP groups compared to C. CONCLUSION: Our results suggest that maternal exercise during pregnancy does not further increase pregnancy-induced cardiac hypertrophy. However, exercise during pregnancy may not be sufficient to activate the genes regulating angiogenesis and the antioxidant defense system.

Korean red ginseng (Panax ginseng) prevents obesity by inhibiting angiogenesis in high fat diet-induced obese C57BL/6J mice

Adipose tissue growth and development are thought to be associated with angiogenesis and extracellular matrix remodeling. Because ginseng has been shown to inhibit angiogenesis and matrix metalloproteinase (MMP) activity, we hypothesized that adipose tissue growth and obesity can be regulated by Korean ginseng (Panax ginseng C.A. Meyer). Wild-type C57BL/6J mice were fed for 8 weeks with a low fat diet, a high fat diet (HFD), or HFD supplemented with 0.5% or 5% Korean red ginseng extract. We measured body weight, adipose tissue mass, food intake, MMP activity, and the expression of genes involved in angiogenesis and MMPs. Administering ginseng to HFD-induced obese mice produced reductions in body weight and adipose tissue mass compared with untreated counterparts. Ginseng treatment decreased blood vessel density and MMP activity in adipose tissues. Ginseng also reduced mRNA levels of angiogenic factors (e.g., VEGF-A and FGF-2) and MMPs (e.g., MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors (e.g., TSP-1, TIMP-1, and TIMP-2) in adipose tissues. These results demonstrate that ginseng effectively reduces adipose tissue mass and prevents obesity in diet-induced obese mice and that this process may be mediated in part through the anti-angiogenic actions of ginseng.

Interrelationship and expression profiling of cyclooxygenase and angiogenic factors in Indian patients with multiple myeloma

Multiple myeloma (MM) is classically illustrated by a desynchronized cytokine system with rise in inflammatory cytokines. There are recent reports which emphasized the potential role of angiogenesis in the development of MM. Role of cyclooxygenase 2 (COX-2) is well documented in the pathogenesis of solid tumors, but little is known about its occurrence and function in hematologic neoplasms. Involvement of neoangiogenesis is reported in the progression of MM, and angiopoietins probably contribute to this progression by enhancing neovascularization. Circulatory and mRNA levels of angiogenic factors and cyclooxygenase were determined in 125 subjects (75 MM patients and 50 healthy controls) by using enzyme-linked immunosorbent assay and quantitative PCR. We observed significant increase for angiogenic factors (Ang-1, Ang-2, hepatocyte growth factor, and vascular endothelial growth factor) and cyclooxygenase at circulatory level, as well as at mRNA level, as compared to healthy controls except insignificant increase for Ang-1 at circulatory level. We have also observed the significant positive correlation of all angiogenic factors with cyclooxygenase. The strong association found between angiogenic factors and COX-2 in this study may lead to the development of combination therapeutic strategy to treat MM. Therefore, targeting COX-2 by using its effective inhibitors demonstrating antiangiogenic and antitumor effects could be used as a new therapeutic approach for treatment of MM.

Compound K, a novel ginsenoside metabolite, inhibits adipocyte differentiation in 3T3-L1 cells: Involvement of angiogenesis and MMPs

Compound K, a novel ginsenoside metabolite formed by intestinal bacteria, is shown to inhibit angiogenesis and matrix metalloproteinase (MMP) activities. Since growth and development of adipose tissue are thought to require adipogenesis, angiogenesis, and extracellular matrix remodeling, we investigated whether compound K inhibits adipocyte differentiation and its potential mechanisms. Treatment of 3T3-L1 adipocytes with compound K inhibited lipid accumulation and expression of adipocyte-specific genes (i.e., PPARγ, leptin, aP2, and C/EBPα). Compound K decreased mRNA levels of angiogenic factors (i.e., VEGF-A and FGF-2) and MMPs (i.e., MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1, and TIMP-2) in 3T3-L1 cells. MMP-2 and MMP-9 activities were also decreased in compound K-treated cells. These results demonstrate that compound K effectively inhibited adipogenesis and that this process may be mediated in part through changes in the expression of genes involved in angiogenesis and MMP system. Thus, by suppressing adipogenesis, compound K likely has therapeutic potential for the treatment of obesity and related disorders.

Reduction of adipose tissue mass by the angiogenesis inhibitor ALS-L1023

Abstract Objective: Angiogenesis has been demonstrated to modulate adipogenesis and obesity. This study was undertaken to determine whether the botanical drug ALS-L1023 (ALS), which exhibits antiangiogenic activity, can regulate adipose tissue growth in high-fat-diet-induced obese mice. Materials and Methods: The effects of ALS on angiogenesis and extracellular matrix remodeling were measured using in vitro assays. The effects of ALS on adipose tissue growth was investigated in nutritionally obese mice. Results: ALS inhibited angiogenesis in a concentration-dependent manner in the tube formation assay and the VEGF-induced proliferation assay using human umbilical vein endothelial cells. ALS also suppressed matrix metalloproteinase (MMP) activity in vitro. ALS administration to high-fat-diet-induced obese mice significantly reduced body weight gain, adipose tissue mass, and adipocyte size compared with the findings in the controls. ALS treatment decreased blood vessel density and MMP activity in adipose tissues. ALS reduced the mRNA levels of angiogenic factors (VEGFs and FGF-2) and MMPs (MMP-2 and MMP-9), whereas it increased the mRNA levels of angiogenic inhibitors (TSP-1 and TIMPs) in adipose tissues. Conclusion: These results suggest that in vivo and in vitro treatment with ALS, which has antiangiogenic and MMP inhibitory activities, reduces adipose tissue mass in nutritionally induced obese mice, providing evidence that adipose tissue growth can be regulated by angiogenesis inhibitors.

The anti-angiogenic herbal extracts Ob-X from Morus alba, Melissa officinalis, and Artemisia capillaris suppresses adipogenesis in 3T3-L1 adipocytes

Context: Growing adipose tissue is thought to require adipogenesis, angiogenesis, and extracellular matrix (ECM) remodeling. Close examination of developing adipose tissue microvasculature reveals that angiogenesis often precedes adipogenesis. Since our previous study demonstrated that Ob-X, the anti-angiogenic herbal composition composed of Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae), reduced adipose tissue mass in obese mice, we hypothesized that adipogenesis can be inhibited by Ob-X.Objective: To investigate the effects of the anti-angiogenic herbal extracts Ob-X on adipogenesis in 3T3-L1 adipocytes.Materials and methods: After differentiated 3T3-L1 adipocytes were treated with Ob-X, we studied the effects of Ob-X on triglyceride accumulation and expression of genes involved in adipogenesis, angiogenesis, and ECM remodeling.Results: Treatment of cells with Ob-X inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or monocyte differentiation-inducing (MDI) mix. Ob-X reduced mRNA levels of angiogenic factors (vascular endothelial growth factor-A, -B, -C, -D, and fibroblast growth factor-2) and matrix metalloproteinases (MMPs; MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors [(thrombospondin-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2)] in differentiated cells. MMP-2 and MMP-9 activities were also decreased in Ob-X-treated cells.Discussion and conclusion: These results suggest that the anti-angiogenic herbal composition Ob-X inhibits differentiation of preadipocytes into adipocytes. These events may be mediated by changes in the expression of genes involved in lipogenesis, angiogenesis, and the MMP system. Thus, by reducing adipogenesis, anti-angiogenic Ob-X provides a possible therapeutic approach for the prevention and treatment of human obesity and its related disorders.

Maternal obesity and overnutrition alter fetal growth rate and cotyledonary vascularity and angiogenic factor expression in the ewe

In pregnant sheep, maternal:fetal exchange occurs across placentomes composed of placental cotyledonary and uterine caruncular tissues. Recently, we reported that fetal weights of obese (OB) ewes [fed a diet of 150% of National Research Council (NRC) recommendations] were approximately 30% greater than those of control (C) ewes (fed a diet 100% of NRC recommendations) at midgestation (MG), but fetal weights were similar in late gestation (LG). Transplacental nutrient exchange is dependent on placental blood flow, which itself is dependent on placental vascularity. The current study investigated whether the observed initial faster and subsequent slower fetal growth rate of OB compared with C was associated with changes in cotyledonary vascularity and expression of angiogenic factors (vascular endothelial growth factor, fibroblast growth factor-2, placental growth factor, angiopoietin-1 and -2). Cotyledonary arteriole diameters were markedly greater (P < 0.05) in OB than C ewes at MG, but while arteriole diameter of C ewes increased (P < 0.05) from MG to LG, they remained unchanged in OB ewes. Cotyledonary arterial angiogenic factors mRNA and protein expression were lower (P < 0.05) in OB than C ewes at MG and remained low from MG to LG. In contrast, mRNA levels of angiogenic factors in C ewes declined from high levels at MG to reach those of OB ewes by LG. The increase in cotyledonary arteriole diameter in early to MG may function to accelerate fetal growth rate in OB ewes, while the decreased cotyledonary arterial angiogenic factors from MG-LG may function to protect the fetus from excessive placental vascular development, increased maternal nutrient delivery, and excessive weight gain.

Fiber capillary regression and endothelial cell apoptosis in the soleus muscle of hindlimb unloaded rats: role of angiogenic regulators

The aim of present study was to determine the changes in the 3‐D architecture of the intramuscular endothelial networks and the responses of angiogenic and its inhibitory factors in atrophied soleus muscles. The hindlimbs of male Wistar rats were unloaded for 2 weeks by tail suspension. The endothelial network of soleus muscle was visualized by confocal microscopy. Apoptotic endothelial cells were identified by DNA fragmentation (TUNEL) using immunofluorescent staining. The mRNA levels of angiogenic factors (VEGF, KDR, Flt‐1, Ang‐1, Ang‐2, Tie‐2, HIF‐1 alpha) and inhibitory factors (TSP‐1 and caspase‐3) were determined by TaqMan probe‐based real‐time PCR. Chronic unloading resulted in a decrease in the number of anastomoses and tortuosity, appearance of TUNEL‐positive endothelial cells, a decrease in angiogenic factor mRNA, an increase in the Ang‐2‐to‐Ang‐1 ratio, and an increase in angiogenic inhibitor mRNA. The data suggest vasculature destabilization with chronic unloading and indicate that both a decrease in angiogenic factors and increase in angiogenic inhibitory factors play a role in this process.Supported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, no. 19300196.

The anti-angiogenic herbal composition Ob-X inhibits adipose tissue growth in obese mice

The growth and development of adipose tissue are thought to be associated with angiogenesis and extracellular matrix remodeling. As the composition of the herbal extract called Ob-X has been shown to have both anti-angiogenic and matrix metalloproteinase (MMP)-inhibiting activities, we hypothesized that growth of adipose tissue can be regulated by Ob-X. The effects of Ob-X on angiogenesis and extracellular matrix remodeling were measured using in vitro and ex vivo assays. The effects of Ob-X on adipose tissue growth were investigated in nutritionally obese mice. Ob-X inhibited angiogenesis in a dose-dependent manner in the human umbilical vein endothelial cell tube formation assay in vitro and the rat aortic ring assay ex vivo. Ob-X also suppressed MMP activity in vitro. Administration of Ob-X to high fat diet-induced obese mice produced significant reductions in body weight gain and adipose tissue mass compared with control. The mass of both visceral (VSC) and subcutaneous (SC) fat was reduced in Ob-X-treated mice. The size of adipocytes in VSC and SC adipose tissues was also significantly reduced in Ob-X-treated mice. Ob-X treatment decreased the blood vessel density and MMP activity in VSC adipose tissues of nutritionally obese mice. Ob-X reduced mRNA levels of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1 and TIMP-2) in SC and VSC adipose tissues of nutritionally obese mice. Ob-X, which has anti-angiogenic and MMP-inhibitory activities, reduces adipose tissue mass in nutritionally induced obese mice, providing evidence that adipose tissue growth and development may be prevented by inhibiting angiogenesis. In addition, these data suggest that regulation of adipose tissue growth by inhibiting angiogenesis may alter the expression of genes involved in angiogenesis and the MMP system.

The anti-angiogenic herbal composition Ob-X inhibits adipose tissue growth in obese mice

Abstract Objective: The growth and development of adipose tissue are thought to be associated with angiogenesis and extracellular matrix remodeling. Since the composition of the herbal extract called Ob-X has been shown to have both anti-angiogenic and matrix metalloproteinase (MMP)-inhibiting activities, we hypothesized that growth of adipose tissue can be regulated by Ob-X. Materials and Methods: The effects of Ob-X on angiogenesis and extracellular matrix remodeling were measured using in vitro and ex vivo assays. The effects of Ob-X on adipose tissue growth were investigated with nutritionally obese mice. Results: Ob-X inhibited angiogenesis in a dose-dependent manner in the human umbilical vein endothelial cell (HUVEC) tube formation assay in vitro and the rat aortic ring assay ex vivo. Ob-X also suppressed MMP activity in vitro. Administration of Ob-X to high fat diet-induced obese mice produced significant reductions in body weight gain and adipose tissue mass, compared to controls. The mass of both subcutaneous (SC) and visceral (VSC) fat was reduced in Ob-X-treated mice. The size of adipocytes in SC and VSC adipose tissues was also significantly reduced in Ob-X-treated mice. Ob-X treatment decreased the blood vessel density and MMP activity in VSC adipose tissues of nutritionally obese mice. Ob-X reduced mRNA levels of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenesis inhibitors (TSP-1 and TIMP-2) in SC and VSC adipose tissues of nutritionally obese mice. Conclusion: Ob-X, which has anti-angiogenic and MMP-inhibitory activities, reduces adipose tissue mass in nutritionally induced obese mice, providing evidence that adipose tissue growth and development may be prevented by inhibiting angiogenesis. In addition, these data suggest that regulation of adipose tissue growth by inhibiting angiogenesis may alter the expression of genes involved in angiogenesis and the MMP system.

Establishment of a new human pancreatic cancer cell line, NOR-P1, with high angiogenic activity and metastatic potential

We present here a new cell line, NOR-P1, established from a metastatic subcutaneous tumor of a patient with pancreatic cancer. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Genetic and molecular analyses revealed high telomerase activity and a mutation in the K- ras oncogene. Of particular interest, the cells express markedly elevated mRNA levels of angiogenic factors, vascular endothelial growth factor and platelet-derived growth factor, as well as other tumor growth-related factors. Subcutaneous transplantation of the NOR-P1 cells into nude mice formed solid, hemorrhagic tumors which were histologically diagnosed as adenocarcinoma with dense blood vessels and severe extravasation of blood. Furthermore, when NOR-P1 cell suspension was injected directly into the pancreas of nude mice, the cells grew rapidly to form intra-pancreatic tumors associated with liver metastases and peritoneal dissemination that resulted in cachexia and subsequent death. These properties suggest that NOR-P1 is an aggressive pancreatic cancer cell line with a high metastatic potential and may serve as a useful experimental model for studying tumor angiogenesis and metastasis of pancreatic cancer.