Change in decorin during aging of rat placenta

By immunohistochemistry, with or without chondroitinases, decorin was found to be distributed in the extracellular matrix of chorionic villi and amnia. The strength of staining intensified with increasing gestational age. Decorin was isolated from the placenta of 13- to 20-day-old pregnant rats and identified by Western blotting, using an antidecorin core protein antibody. The molecular weight of decorin is approximately 100 kDa, whereas the respective figures for the core protein treated with chondroitinase (chase) ABC and with chase B are approximately 40 kDa and 43 kDa. The difference in the molecular weight between the core protein with chase ABC and B suggests that the glycosaminoglycan (GAG)- base structure on the core protein was chondroitin sulfate (CS) without dermatan sulfate (DS). The decorin content and the proportion of CS to DS in GAG increased with age. We concluded that the age-related changes in the GAG chain may be related to specific functional properties and may have a crucial role in placental tissue organization.     

Gamma‐aminobutyric acidA receptor agonist,muscimol, increases KiSS‐1 gene expression in hypothalamic cell models

Purpose

Accumulating evidence indicates that hypothalamic kisspeptin plays a pivotal role in the regulation of the hypothalamic–pituitary–gonadal (HPG) axis. In this study, the direct action of the gamma‐aminobutyric acid (GABA)_A receptor agonist on kisspeptin‐expressing neuronal cells was examined.

Methods

A hypothalamic cell model of rat hypothalamic cell line R8 (rHypoE8) cells and primary cultures of neuronal cells from fetal rat brains were stimulated with a potent and selective GABA_A receptor agonist, muscimol, to determine the expression of the KiSS‐1 gene.

Results

Stimulation of the rHypoE8 cells with muscimol significantly increased the level of KiSS‐1 messenger (m)RNA expression. The ability of muscimol to increase the level of KiSS‐1 mRNA also was observed in the primary cultures of the neuronal cells from the fetal rat brains. The muscimol‐induced increase in KiSS‐1 mRNA expression was completely inhibited in the presence of the GABA_A receptor antagonist. Although muscimol increased the expression of KiSS‐1, the natural compound, GABA, failed to induce the expression of KiSS‐1 in the rHypoE8 cells. Muscimol did not modulate gonadotropin‐releasing hormone expression in either the rHypoE8 cells or the primary cultures of the fetal rat brains.

Conclusions

This study's observations suggest that the activation of the GABA_A receptor modulates the HPG axis by increasing kisspeptin expression in the hypothalamic neurons.     

Intracavernous Delivery of Freshly Isolated Stromal Vascular Fraction Rescues Erectile Function by Enhancing Endothelial Regeneration in the Streptozotocin‐Induced Diabetic Mouse

IntroductionMen with diabetic erectile dysfunction (ED) often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase‐5 inhibitors.AimTo examine whether and how freshly isolated stromal vascular fraction (SVF) promotes cavernous endothelial regeneration and restores erectile function in diabetic animals.MethodsEight‐week‐old C57BL/6J mice were used. Diabetes was induced by intraperitoneal injection of streptozotocin. SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. At 8 weeks after the induction of diabetes, the animals were divided into six groups: controls, diabetic mice, and diabetic mice treated with a single intracavernous injection of phosphate‐buffered saline (PBS) or SVF (1 × 10⁴ cells, 1 × 10⁵ cells, or 2 × 10⁵ cells/20 µL, respectively).Main Outcome MeasuresTwo weeks later, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to CD31, CD34, phosphohistone H3, phospho‐endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor‐A (VEGF‐A). We also performed Western blot for phospho‐eNOS and eNOS, and determined cyclic guanosine monophosphate (cGMP) concentration in the corpus cavernosum tissue.ResultsSignificant improvement in erectile function was noted in diabetic mice treated with SVF at concentrations of 1 × 10⁵ and 2 × 10⁵ cells, which reached up to 82% of the control values. Local delivery of SVF significantly increased cavernous endothelial cell proliferation, eNOS phosphorylation, and cGMP expression compared with that in the untreated group and the PBS‐treated diabetic group. Intracavernous injection of SVF increased cavernous VEGF‐A expression and induced recruitment of CD34(+)CD31(?) progenitor cells. Some SVF underwent differentiation into cavernous endothelial cells. SVF‐induced promotion of cavernous angiogenesis and erectile function was abolished in the presence of VEGF‐Trap, a soluble VEGF‐A neutralizing antibody.ConclusionThe results support the concept of cavernous endothelial regeneration by use of SVF as a curative therapy for diabetic ED. Ryu J‐K, Tumurbaatar M, Jin H‐R, Kim WJ, Kwon M‐H, Piao S, Choi MJ, Yin GN, Song K‐M, Kang Y‐J, Koh YJ, Koh GY, and Suh J‐K. Intracavernous delivery of freshly isolated stromal vascular fraction rescues erectile function by enhancing endothelial regeneration in the streptozotocin‐induced diabetic mouse. J Sex Med 2012;9:3051–3065.     

Intracavernous Delivery of Synthetic Angiopoietin-1 Protein as a Novel Therapeutic Strategy for Erectile Dysfunction in the Type II Diabetic db/db Mouse

IntroductionPatients with erectile dysfunction (ED) associated with type II diabetes often have impaired endothelial function and tend to respond poorly to oral phosphodiesterase type 5 inhibitors. Therefore, neovascularization is a promising strategy for curing diabetic ED.AimTo determine the effectiveness of a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous angiogenesis and erectile function in a mouse model of type II diabetic ED.MethodsSixteen-week-old male db/db mice (in which obesity and type II diabetes are caused by a mutation in the leptin receptor) and control C57BL/6J mice were used and divided into four groups (N = 14 per group): age-matched controls; db/db mice receiving two successive intracavernous injections of phosphate-buffered saline (PBS) (days ?3 and 0; 20 µL); db/db mice receiving a single intracavernous injection of COMP-Ang1 protein (day 0; 5.8 µg/20 µL); and db/db mice receiving two successive intracavernous injections of COMP-Ang1 protein (days ?3 and 0; 5.8 µg/20 µL).Main Outcome MeasuresTwo weeks later, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with antibodies to platelet/endothelial cell adhesion molecule-1 (PECAM-1) (endothelial cell marker), phosphohistone H3 (PH3, a nuclear protein indicative of cell proliferation), phospho-endothelial nitric oxide synthase (eNOS), and eNOS. Penis specimens from a separate group of animals were used for cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) quantification.ResultsLocal delivery of COMP-Ang1 protein significantly increased eNOS phosphorylation and cGMP and cAMP expression compared with that in the group treated with PBS. Repeated intracavernous injections of COMP-Ang1 protein completely restored erectile function and cavernous endothelial content through enhanced cavernous neoangiogenesis as evaluated by PECAM-1 and PH3 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, whereas a single injection of COMP-Ang1 protein elicited partial improvement.ConclusionCavernous neovascularization using recombinant Ang1 protein is a novel therapeutic strategy for the treatment of ED resulting from type II diabetes. Jin H-R, Kim WJ, Song JS, Piao S, Tumurbaatar M, Shin SH, Choi MJ, Tuvshintur B, Song K-M, Kwon M-H, Yin GN, Koh GY, Ryu J-K, and Suh J-K. Intracavernous delivery of synthetic angiopoietin-1 protein as a novel therapeutic strategy for erectile dysfunction in the type II diabetic db/db mouse.     

Intracavernous delivery of a designed angiopoietin-1 variant rescues erectile function by enhancing endothelial regeneration in the streptozotocin-induced diabetic mouse

OBJECTIVE

Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals.

RESEARCH DESIGN AND METHODS

Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days −3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test.

RESULTS

Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47^phox and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1–treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1–induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin.

CONCLUSIONS

These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as a curative therapy for diabetic erectile dysfunction.Erectile dysfunction affects up to 75% of all men with diabetes and occurs earlier in such patients than in the general population (1,2). The Massachusetts Male Aging Study revealed that erectile dysfunction is three times as prevalent in diabetic men as in men without diabetes (3). Moreover, men with diabetic erectile dysfunction tend to respond less positively to the currently available oral phosphodiesterase-5 (PDE5) inhibitors than do nondiabetic men (4–6). The reduced responsiveness to PDE5 inhibitors in patients with diabetes may be related to the severity of endothelial dysfunction (7). Because the effects of PDE5 inhibitors rely on endogenous nitric oxide (NO) formation, PDE5 inhibitors may fail to increase the level of cyclic guanosine monophosphate (cGMP) above the necessary threshold if the bioavailable NO is insufficient as a result of severe endothelial dysfunction. Furthermore, PDE5 inhibitors must be used on demand, thus limiting the spontaneity of the sexual act. The use of PDE5 inhibitors is absolutely contraindicated in men who take nitrate compounds because of the risk of extreme hypotension or even death (8). Therefore, new therapeutic strategies are needed.Augmented generation of reactive oxygen species (ROS) is one of the major causes of decreased NO bioavailability in diabetes (9–12). We recently reported in streptozotocin (STZ)-induced type 1 diabetic mice that increased superoxide anion production and peroxynitrite formation in the corpus cavernosum tissue play an important role in the diabetes-induced functional and structural impairments in the cavernous endothelium, such as the decrease in endothelial NO synthase (eNOS) enzyme activity, endothelial cell apoptosis, and subsequent loss of endothelial cell content (12). Therefore, restoring NO bioavailability by inhibiting ROS-mediated endothelial cell damage, regenerating cavernous endothelial cells, or both may serve as promising therapeutic strategies for treating patients with diabetic erectile dysfunction.Various antioxidants have been shown to improve endothelial function and erectile ability in animal models of diabetic erectile dysfunction (10,13,14). Adenovirus-mediated gene transfer of extracellular superoxide dismutase into the corpus cavernosum tissue was shown to reduce superoxide anion levels and raise cavernous cGMP levels, which resulted in partial recovery of erectile function in STZ-induced diabetic rats 2 days after transfection (14). Despite encouraging results in preclinical studies, however, no antioxidants have so far been approved for the treatment of patients with erectile dysfunction. A possible reason is that antioxidant therapy may not be effective if structural damage to cavernous endothelial cells has already progressed. In this regard, regeneration of damaged endothelial cells using angiogenic factors is a more logical approach to treating erectile dysfunction than is the prevention of endothelial cell damage with antioxidants.Local delivery of the vascular endothelial growth factor-A (VEGF-A) gene or protein into the penis has been shown to induce partial or complete recovery of erectile function in STZ-induced type 1 diabetic rats (15–17). However, these studies did not demonstrate whether VEGF-A therapy induces endothelial regeneration. Furthermore, VEGF-A often leads to the formation of disorganized vessels that are leaky, hyperpermeable, and inflamed in experimental systems (18,19), thus greatly compromising the therapeutic utility of VEGF-A. In comparison, angiopoietin-1 (Ang1), the ligand of the Tie2 receptor tyrosine kinase, is a specific growth factor that functions to generate a nonleaky, stable, and functional vasculature (19–22). Transgenic overexpression or gene transfer of Ang1 not only enhances vessel formation but also protects the adult vasculature against vascular leakage (19,23–25). Thus, Ang1 has potential therapeutic applications in angiogenesis and the prevention of vascular leakage. However, our previous study (26) revealed that a single intracavernous delivery of the adenovirus-mediated Ang1 gene failed to induce an angiogenic response in the penis of a hypercholesterolemic rat. Recently, Cho et al. (27) developed a soluble and potent Ang1 variant, cartilage oligomeric matrix protein (COMP)-Ang1, that is more potent than native Ang1 in phosphorylating Tie2 in primary cultured endothelial cells. COMP-Ang1 stimulates angiogenesis with nonleaky neovessel formation in the mouse corneal micropocket assay, whereas VEGF-A stimulates angiogenesis with leaky neovessel formation (27). One mechanism for inducing nonleaky and healthy angiogenesis is the specific activation of Tie2 in endothelial cell-cell or cell-matrix contacts by COMP-Ang1 (28,29).In the current study, we determined the effectiveness of COMP-Ang1 in promoting cavernous endothelial regeneration and restoring erectile function in a mouse model of diabetic erectile dysfunction. In addition, because increased vascular permeability by loss of endothelial cell-cell junction proteins is an important pathophysiological mechanism involved in diabetic retinopathy (30–33), we investigated whether COMP-Ang1 induces nonleaky angiogenesis in the penis by restoring endothelial cell-cell junction proteins.     

Increased incidence of aflatoxin B1-induced liver tumors in hepatitis virus C transgenic mice

Viral hepatitis and aflatoxin B1 (AFB1) exposure are common risk factors for hepatocellular carcinoma (HCC). The incidence of HCC in individuals coexposed to hepatitis C (HCV) or B virus and AFB1 is greater than could be explained by the additive effect; yet, the mechanisms are poorly understood because of the lack of an animal model. Our study investigated the outcomes and mechanisms of combined exposure to HCV and AFB1. We hypothesized that HCV transgenic (HCV-Tg; expressing core, E1, E2 and p7, nucleotides 342-2771) mice will be prone to hepatocarcinogenesis when exposed to AFB1. Neonatal (7 days old) HCV-Tg or C57BL/6J wild-type (WT) mice were exposed to AFB1 (6 μg/g bw) or tricaprylin vehicle (15 μl/g bw), and male offspring were followed for up to 12 months. No liver lesions were observed in vehicle-treated WT or HCV-Tg mice. Tumors (adenomas or carcinomas) and preneoplastic lesions (hyperplasia or foci) were observed in 22.5% (9 of 40) of AFB1-treated WT mice. In AFB1-treated HCV-Tg mice, the incidence of tumorous or pretumorous lesions was significantly elevated (50%, 18 of 36), with the difference largely due to a 2.5-fold increase in the incidence of adenomas (30.5 vs. 12.5%). Although oxidative stress and steatohepatitis were observed in both AFB1-treated groups, molecular changes indicative of the enhanced inflammatory response and altered lipid metabolism were more pronounced in HCV-Tg mice. In summary, HCV proteins core, E1, E2 and p7 are sufficient to reproduce the cocarcinogenic effect of HCV and AFB1, which is a known clinical phenomenon.