Sildenafil-Induced Revascularization of Rat Hindlimb Involves Arteriogenesis through PI3K/AKT and eNOS Activation

Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil–treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.     

Effect of Guibi-Tang,a Traditional Herbal Formula,on Retinal Neovascularization in a Mouse Model of Proliferative Retinopathy

Ocular pathologic angiogenesis is an important causative risk factor of blindness in retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular macular degeneration. Guibi-tang (GBT) is a frequently used oriental herbal formula in East Asian countries, and is also called Qui-pi-tang in Chinese and Kihi-To in Japanese. In the present study, we investigated the preventive effect of GBT on retinal pathogenic neovascularization in a mouse model of oxygen-induced retinopathy (OIR). C57BL/6 mice were exposed to 75% hyperoxia for five days on postnatal day 7 (P7). The mice were then exposed to room air from P12 to P17 to induce ischemic proliferative retinopathy. GBT (50 or 100 mg/kg/day) was intraperitoneally administered daily for five days (from P12 to P16). On P17, Retinal neovascularization was measured on P17, and the expression levels of 55 angiogenesis-related factors were analyzed using protein arrays. GBT significantly decreased retinal pathogenic angiogenesis in OIR mice, and protein arrays revealed that GBT decreased PAI-1 protein expression levels. Quantitative real-time PCR revealed that GBT reduced vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and plasminogen activator inhibitor 1 (PAI-1) mRNA levels in OIR mice. GBT promotes potent inhibitory activity for retinal neovascularization by decreasing VEGF, FGF2, and PAI-1 levels.     

Soluble Epoxide Hydrolase in Aged Female Mice and Human Explanted Hearts Following Ischemic Injury

Myocardial infarction (MI) accounts for a significant proportion of death and morbidity in aged individuals. The risk for MI in females increases as they enter the peri-menopausal period, generally occurring in middle-age. Cytochrome (CYP) 450 metabolizes N-3 and N-6 polyunsaturated fatty acids (PUFA) into numerous lipid mediators, oxylipids, which are further metabolised by soluble epoxide hydrolase (sEH), reducing their activity. The objective of this study was to characterize oxylipid metabolism in the left ventricle (LV) following ischemic injury in females. Human LV specimens were procured from female patients with ischemic cardiomyopathy (ICM) or non-failing controls (NFC). Female C57BL6 (WT) and sEH null mice averaging 13–16 months old underwent permanent occlusion of the left anterior descending coronary artery (LAD) to induce myocardial infarction. WT (wild type) mice received vehicle or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB), in their drinking water ad libitum for 28 days. Cardiac function was assessed using echocardiography and electrocardiogram. Protein expression was determined using immunoblotting, mitochondrial activity by spectrophotometry, and cardiac fibre respiration was measured using a Clark-type electrode. A full metabolite profile was determined by LC–MS/MS. sEH was significantly elevated in ischemic LV specimens from patients, associated with fundamental changes in oxylipid metabolite formation and significant decreases in mitochondrial enzymatic function. In mice, pre-treatment with tAUCB or genetic deletion of sEH significantly improved survival, preserved cardiac function, and maintained mitochondrial quality following MI in female mice. These data indicate that sEH may be a relevant pharmacologic target for women with MI. Although future studies are needed to determine the mechanisms, in this pilot study we suggest targeting sEH may be an effective strategy for reducing ischemic injury and mortality in middle-aged females.     

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE−/− Mice

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE^−/−) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE^−/−/S1PR3^−/− mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.     

小鼠Sca-1^+干细胞对心肌梗死模型小鼠的疗效及其基因调控机制

目的探讨小鼠干细胞抗原-1阳性(stem cell antigen-1-positive,Sca-1^+)干细胞(stem cells,SCs)对心肌梗死(myocardial infarction,MI)模型小鼠的疗效及其基因调控机制。方法采用免疫磁珠分选法急性分离2日龄及3、6、9、12月龄小鼠心脏Sca-1^+SCs;通过结扎8周龄小鼠心脏冠状动脉左主降支,建立小鼠急性MI模型,于心梗边缘区分别注射PBS和Sca-1^+SCs悬液。术后行超声心动图测定心功能,Masson三色染色、心重/体重比(HW/BW)用于评价各组Sca-1^+SCs对心脏结构的影响。将各组小鼠心脏Sca-1^+SCs进行全基因组测序,通过STEM软件对转录组基因表达水平进行聚类及GO富集分析,筛选出乳鼠和成年鼠差异表达的基因类型,并采用qRT-PCR法进一步验证目的基因的表达情况。结果 MI术后,与PBS组比较,乳鼠心脏Sca-1^+SCs治疗组小鼠左室射血分数(left ventricular ejection fraction,LVEF)和左室短轴缩短率(left ventricular fraction shortening,LVFS)均明显升高(P <0. 05),HW/BW明显降低(P <0. 05),左室舒张末内径(left ventricular diastolic internal diameter,LVIDd)明显减小(P <0. 05);与成年鼠比较,乳鼠心脏Sca-1^+SCs移植治疗可降低心肌梗死面积,缓解心腔扩大。在乳鼠心脏Sca-1^+SCs中高表达,而成年鼠表达降低的基因共185个,regulation of developmental process条目上富集基因有38个,其中肌球蛋白重链6(myosin heavy chain6 cardiac muscle alpha,Myh6)、胰岛素样生长因子2(insulin-like growth factor 2,Igf2)、成骨细胞特异性因子(periostin,Postn)、神经元再生相关蛋白(neuronal regeneration related protein,Nrep)基因在乳鼠心脏Sca-1^+SCs中具有较高转录水平。与乳鼠比较,成年小鼠心脏Sca-1^+SCs中Myh6、Igf2、Postn、Nrep基因m RNA的表达水平均显著下调(P <0. 05)。结论乳鼠心脏Sca-1^+SCs移植对MI的疗效优于成年小鼠,其原因可能是乳鼠心脏Sca-1^+SCs中Myh6、Igf2、Postn、Nrep等基因的表达水平高于成年小鼠。     

Nourin-Associated miRNAs: Novel Inflammatory Monitoring Markers for Cyclocreatine Phosphate Therapy in Heart Failure

Background: Cyclocreatine phosphate (CCrP) is a potent bioenergetic cardioprotective compound known to preserve high levels of cellular adenosine triphosphate during ischemia. Using the standard Isoproterenol (ISO) rat model of heart failure (HF), we recently demonstrated that the administration of CCrP prevented the development of HF by markedly reducing cardiac remodeling (fibrosis and collagen deposition) and maintaining normal ejection fraction and heart weight, as well as physical activity. The novel inflammatory mediator, Nourin is a 3-KDa formyl peptide rapidly released by ischemic myocardium and is associated with post-ischemic cardiac inflammation. We reported that the Nourin-associated miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) are significantly upregulated in unstable angina patients and patients with acute myocardial infarction, but not in healthy subjects. Objectives: To test the hypothesis that Nourin-associated miR-137 and miR-106b-5p are upregulated in ISO-induced “HF rats” and that the administration of CCrP prevents myocardial injury (MI) and reduces Nourin gene expression in “non-HF rats”. Methods: 25 male Wistar rats (180–220 g) were used: ISO/saline (n = 6), ISO/CCrP (0.8 g/kg/day) (n = 5), control/saline (n = 5), and control/CCrP (0.8 g/kg/day) (n = 4). In a limited study, CCrP at a lower dose of 0.4 g/kg/day (n = 3) and a higher dose of 1.2 g/kg/day (n = 2) were also tested. The Rats were injected SC with ISO for two consecutive days at doses of 85 and 170 mg/kg/day, respectively, then allowed to survive for an additional two weeks. CCrP and saline were injected IP (1 mL) 24 h and 1 h before first ISO administration, then daily for two weeks. Serum CK-MB (U/L) was measured 24 h after the second ISO injection to confirm myocardial injury. After 14 days, gene expression levels of miR-137 and miR-106b-5p were measured in serum samples using quantitative real-time PCR (qPCR). Results: While high levels of CK-MB were detected after 24 h in the ISO/saline rats indicative of MI, the ISO/CCrP rats showed normal CK-MB levels, supporting prevention of MI by CCrP. After 14 days, gene expression profiles showed significant upregulation of miR-137 and miR-106b-5p by 8.6-fold and 8.7-fold increase, respectively, in the ISO/saline rats, “HF rats,” compared to the control/saline group. On the contrary, CCrP treatment at 0.8 g/kg/day markedly reduced gene expression of miR-137 by 75% and of miR-106b-5p by 44% in the ISO/CCrP rats, “non-HF rats,” compared to the ISO/Saline rats, “HF rats.” Additionally, healthy rats treated with CCrP for 14 days showed no toxicity in heart, liver, and renal function. Conclusions: Results suggest a role of Nourin-associated miR-137 and miR-106b-5p in the pathogenesis of HF and that CCrP treatment prevented ischemic injury in “non-HF rats” and significantly reduced Nourin gene expression levels in a dose–response manner. The Nourin gene-based mRNAs may, therefore, potentially be used as monitoring markers of drug therapy response in HF, and CCrP—as a novel preventive therapy of HF due to ischemia.     

Sphingosine 1-Phosphate Receptor 5 (S1P5) Knockout Ameliorates Adenine-Induced Nephropathy

S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P₅ has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P₅ in renal inflammation and fibrosis. Male S1P₅ knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P₅ knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P₅ knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P₅ might be a promising goal for the pharmacological treatment of kidney diseases.     

Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles

Avascular transplantation of frozen–thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia–reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4–5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin–eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.     

The Course of AαVal541 as a Proteinase 3 Specific Neo-Epitope after Alpha-1-Antitrypsin Augmentation in Severe Deficient Patients

In alpha-1-antitrypsin deficiency (AATD), neutrophil serine proteases such as elastase and proteinase 3 (PR3) are insufficiently inhibited. A previous study in AATD patients showed a higher plasma level of the specific PR3-generated fibrinogen-derived peptide AαVal541, compared with healthy controls. Here, we analyzed the course of AαVal541 plasma levels during 4 weeks after a single iv dose of 240 mg/kg AAT in ten patients with genotype Z/Rare or Rare/Rare. To this end, we developed an immunoassay to measure AαVal541 in plasma and applied population pharmacokinetic modeling for AAT. The median AαVal541 plasma level before treatment was 140.2 nM (IQR 51.5–234.8 nM)). In five patients who received AAT for the first time, AαVal541 levels decreased to 20.6 nM (IQR 5.8–88.9 nM), and in five patients who already had received multiple infusions before, it decreased to 26.2 nM (IQR 22.31–35.0 nM). In 9 of 10 patients, AαVal541 levels were reduced to the median level of healthy controls (21.4 nM; IQR 16.7–30.1 nM). At 7–14 days after treatment, AαVal541 levels started to increase again in all patients. Our results show that fibrinopeptide AαVal541 may serve as a biochemical footprint to assess the efficacy of in vivo inhibition of PR3 activity in patients receiving intravenous AAT augmentation therapy.     

Sodium Hydrosulfide Prevents Myocardial Dysfunction through Modulation of Extracellular Matrix Accumulation and Vascular Density

The aim was to examine the role of exogenous hydrogen sulfide (H₂S) on cardiac remodeling in post-myocardial infarction (MI) rats. MI was induced in rats by ligation of coronary artery. After treatment with sodium hydrosulfide (NaHS, an exogenous H₂S donor, 56 μM/kg·day) for 42 days, the effects of NaHS on left ventricular morphometric features, echocardiographic parameters, heme oxygenase-1 (HO-1), matrix metalloproteinases-9 (MMP-9), type I and type III collagen, vascular endothelial growth factor (VEGF), CD34, and α-smooth muscle actin (α-SMA) in the border zone of infarct area were analyzed to elucidate the protective mechanisms of exogenous H₂S on cardiac function and fibrosis. Forty-two days post MI, NaHS-treatment resulted in a decrease in myocardial fibrotic area in association with decreased levels of type I, type III collagen and MMP-9 and improved cardiac function. Meanwhile, NaHS administration significantly increased cystathionine γ-lyase (CSE), HO-1, α-SMA, and VEGF expression. This effect was accompanied by an increase in vascular density in the border zone of infarcted myocardium. Our results provided the strong evidences that exogenous H₂S prevented cardiac remodeling, at least in part, through inhibition of extracellular matrix accumulation and increase in vascular density.     

n-3 Polyunsaturated Fatty Acids Improve Inflammation via Inhibiting Sphingosine Kinase 1 in a Rat Model of Parenteral Nutrition and CLP-Induced Sepsis

The sphingosine kinase 1 (SphK1)/sphingosine‐1‐phosphate (S1P) pathway plays a key role in inflammation. Parenteral nutrition containing n‐3 polyunsaturated fatty acids (n‐3 PUFA) may regulate inflammatory reactions. The aim of this study is to determine whether n‐3 PUFA may improve inflammatory responses by neutralizing SphK1 signaling. Rat models of parenteral nutrition, cecal ligation and puncture (CLP)‐induced sepsis were generated. Male Sprague–Dawley rats were operated for CLP on day 2 after venous catheterization. The rats were randomized to receive normal saline (NS; n = 20), parenteral nutrition (PN; n = 20), or PN + fish oil (FO; n = 20) for 5 days. The daily intake of fish oil (1.25–2.82 g EPA and 1.44–3.09 g DHA per 100 ml) in the FO group was approximately 1.8 g/kg body weight/day. Rats in the control group (n = 10) were subjected to sham operation and received a chow diet. Spleen tissues were collected for SphK1 and S1P receptor expression analysis. Our data showed that n‐3 PUFA ameliorated the survival rate. SphK1 expression and its enzymatic activity were significantly upregulated in sepsis rats. Furthermore, mRNA and protein levels of S1PR3, but not S1PR1, were also facilitated after CLP. However, PN + FO dramatically decreased SphK1 mRNA level and its enzymatic activity. S1PR3 expression was also attenuated by FO addition. In conclusion, the anti‐inflammatory effect of n‐3 PUFA may be linked to the inhibition of the SphK1/S1P pathway in a rat model of parenteral nutrition and CLP‐induced sepsis.     

Endogenous Protease Nexin-1 Protects against Cerebral Ischemia

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin’s endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced β-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1^−/− mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.     

Focal Ischemic Injury to the Early Neonatal Rat Brain Models Cognitive and Motor Deficits with Associated Histopathological Outcomes Relevant to Human Neonatal Brain Injury

Neonatal arterial ischemic stroke is one of the more severe birth complications. The injury can result in extensive neurological damage and is robustly associated with later diagnoses of cerebral palsy (CP). An important part of efforts to develop new therapies include the on-going refinement and understanding of animal models that capture relevant clinical features of neonatal brain injury leading to CP. The potent vasoconstrictor peptide, Endothelin-1 (ET-1), has previously been utilised in animal models to reduce local blood flow to levels that mimic ischemic stroke. Our previous work in this area has shown that it is an effective and technically simple approach for modelling ischemic injury at very early neonatal ages, resulting in stable deficits in motor function. Here, we aimed to extend this model to also examine the impact on cognitive function. We show that focal delivery of ET-1 to the cortex of Sprague Dawley rats on postnatal day 0 (P0) resulted in impaired learning in a touchscreen-based test of visual discrimination and correlated with important clinical features of CP including damage to large white matter structures.     

Impairment of IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity and Mortality

Following exposure to high doses of ionizing radiation, diverse strains of vertebrate species will manifest varying levels of radiation sensitivity. To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, two mouse strains with varying radiosensitivity (C3H/HeN, and CD2F1), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling pathway is associated with radiosensitivity, we investigated the link between systemic or tissue-specific IGF-1 signaling and radiosensitivity. Adult male C3H/HeN and CD2F1 mice were irradiated using gamma photons at Lethal Dose-70/30 (LD_70/30), 7.8 and 9.35 Gy doses, respectively. Those mice that survived up to 30 days post-irradiation, were termed the survivors. Mice that were euthanized prior to 30 days post-irradiation due to deteriorated health were termed decedents. The analysis of non-irradiated and irradiated survivor and decedent mice showed that inter-strain radiosensitivity and post-irradiation survival outcomes are associated with activation status of tissue and systemic IGF-1 signaling, nuclear factor erythroid 2–related factor 2 (Nrf2) activation, and the gene expression profile of cardiac mitochondrial energy metabolism pathways. Our findings link radiosensitivity with dysregulation of IGF-1 signaling, and highlight the role of antioxidant gene response and mitochondrial function in radiation sensitivity.     

Etoposide-Entrapped Progesterone-Cationic Lipid Nanoaggregates as Selective Therapeutics against Etoposide-Resistant Colorectal Cancer Cells

Drug resistance has a major impact on the treatment of several cancers. This is mainly due to the overexpression of cellular drug efflux proteins. Hence, drug-delivery systems that can avoid this resistance are needed. We report PR10, a progesterone-cationic lipid conjugate, as a self-assembling nanoaggregate that delivers a drug cargo of etoposide, a topoisomerase inhibitor, selectively to cancer cells. In this study, we observed that etoposide nanoaggregates (P : E) caused selective and enhanced toxicity in etoposide-resistant CT26 cancer cells (IC₅₀ 9 μM) compared to when etoposide (IC₅₀>20 μM) was used alone. Concurrently, no toxicity was observed in etoposide-sensitive HEK293 cells for P : E treatment (IC₅₀>20 μM). The P : E-treated cancer cells seem to have no effect on ABCB1 expression, but etoposide-treated cells exhibited a twofold increase in ABCB1 expression, a potent efflux protein for several xenobiotic compounds. This observation supports the notion that the enhanced toxicity of P : E nanoaggregates is due to their ability to keep the expression of ABCB1 low, thus allowing longer intracellular residence of etoposide. In a BALB/c orthotopic colorectal cancer model, the nanoaggregates led to enhanced survival (45 days) compared to etoposide-treated mice (39 days). These findings suggest that PR10 could be used as a potential cancer-selective etoposide delivery vehicle to treat several etoposide-resistant cancers with fewer side effects due to the nonspecific toxicity of the drug.     

Antiangiogenesis,Loss of Cell Adhesion and Apoptosis Are Involved in the Antitumoral Activity of Proteases from V. cundinamarcensis (C. candamarcensis) in Murine Melanoma B16F1

The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-β displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.     

Metformin Attenuates Postinfarction Myocardial Fibrosis and Inflammation in Mice

Diabetes is a major risk factor for the development of cardiovascular disease with a higher incidence of myocardial infarction. This study explores the role of metformin, a first-line antihyperglycemic agent, in postinfarction fibrotic and inflammatory remodeling in mice. Three-month-old C57BI/6J mice were submitted to 30 min cardiac ischemia followed by reperfusion for 14 days. Intraperitoneal treatment with metformin (5 mg/kg) was initiated 15 min after the onset of reperfusion and maintained for 14 days. Real-time PCR was used to determine the levels of COL3A1, αSMA, CD68, TNF-α and IL-6. Increased collagen deposition and infiltration of macrophages in heart tissues are associated with upregulation of the inflammation-associated genes in mice after 14 days of reperfusion. Metformin treatment markedly reduced postinfarction fibrotic remodeling and CD68-positive cell population in mice. Moreover, metformin resulted in reduced expression of COL3A1, αSMA and CD68 after 14 days of reperfusion. Taken together, these results open new perspectives for the use of metformin as a drug that counteracts adverse myocardial fibroticand inflammatory remodeling after MI.     

The Effect of Sodium-Dependent Glucose Cotransporter 2 Inhibitor Tofogliflozin on Neurovascular Coupling in the Retina in Type 2 Diabetic Mice

We investigated the effect of tofogliflozin, a sodium-dependent glucose cotransporter 2 inhibitor (SGLT2i), on retinal blood flow dysregulation, neural retinal dysfunction, and the impaired neurovascular coupling in type 2 diabetic mice. Tofogliflozin was added to mouse chow to deliver 5 mg/kg/day and 6-week-old mice were fed for 8 weeks. The longitudinal changes in the retinal neuronal function and blood flow responses to systemic hyperoxia and flicker stimulation were evaluated every 2 weeks in diabetic db/db mice that received tofogliflozin (n =6) or placebo (n = 6) from 8 to 14 weeks of age. We also evaluated glial activation and vascular endothelial growth factor (VEGF) expression by immunofluorescence. Tofogliflozin treatment caused a sustained decrease in blood glucose in db/db mice from 8 weeks of the treatment. In tofogliflozin-treated db/db mice, both responses improved from 8 to 14 weeks of age, compared with vehicle-treated diabetic mice. Subsequently, the electroretinography implicit time for the oscillatory potential was significantly improved in SGLT2i-treated db/db mice. The systemic tofogliflozin treatment prevented the activation of glial fibrillary acidic protein and VEGF protein expression, as detected by immunofluorescence. Our results suggest that glycemic control with tofogliflozin significantly improved the impaired retinal neurovascular coupling in type 2 diabetic mice with the inhibition of retinal glial activation.     

Cerebral Hyperperfusion after Revascularization Inhibits Development of Cerebral Ischemic Lesions Due to Artery-to-Artery Emboli during Carotid Exposure in Endarterectomy for Patients with Preoperative Cerebral Hemodynamic Insufficiency: Revisiting the “Impaired Clearance of Emboli” Concept

The purpose of the present study was to determine whether cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to artery-to-artery emboli during exposure of the carotid arteries in carotid endarterectomy (CEA). In patients undergoing CEA for internal carotid artery stenosis (≥70%), cerebral blood flow (CBF) was measured using single-photon emission computed tomography (SPECT) before and immediately after CEA. Microembolic signals (MES) were identified using transcranial Doppler during carotid exposure. Diffusion-weighted magnetic resonance imaging (DWI) was performed within 24 h after surgery. Of 32 patients with a combination of reduced cerebrovascular reactivity to acetazolamide on preoperative brain perfusion SPECT and MES during carotid exposure, 14 (44%) showed cerebral hyperperfusion (defined as postoperative CBF increase ≥100% compared with preoperative values), and 16 (50%) developed DWI-characterized postoperative cerebral ischemic lesions. Postoperative cerebral hyperperfusion was significantly associated with the absence of DWI-characterized postoperative cerebral ischemic lesions (95% confidence interval, 0.001–0.179; p = 0.0009). These data suggest that cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to artery-to-artery emboli during carotid exposure in CEA, supporting the “impaired clearance of emboli” concept. Blood pressure elevation following carotid declamping would be effective when embolism not accompanied by cerebral hyperperfusion occurs during CEA.     

Sphingosine-1-Phosphate-Triggered Expression of Cathelicidin LL-37 Promotes the Growth of Human Bladder Cancer Cells

It has been proven that tumour growth and progression are regulated by a variety of mediators released during the inflammatory process preceding the tumour appearance, but the role of inflammation in the development of bladder cancer is ambiguous. This study was designed around the hypothesis that sphingosine-1-phosphate (S1P), as a regulator of several cellular processes important in both inflammation and cancer development, may exert some of the pro-tumorigenic effects indirectly due to its ability to regulate the expression of human cathelicidin (hCAP-18). LL-37 peptide released from hCAP-18 is involved in the development of various types of cancer in humans, especially those associated with infections. Using immunohistological staining, we showed high expression of hCAP-18/LL-37 and sphingosine kinase 1 (the enzyme that forms S1P from sphingosine) in human bladder cancer cells. In a cell culture model, S1P was able to stimulate the expression and release of hCAP-18/LL-37 from human bladder cells, and the addition of LL-37 peptide dose-dependently increased their proliferation. Additionally, the effect of S1P on LL-37 release was inhibited in the presence of FTY720P, a synthetic immunosuppressant that blocks S1P receptors. Together, this study presents the possibility of paracrine relation in which LL-37 production following cell stimulation by S1P promotes the development and growth of bladder cancer.