Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice.

Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+) (500,000 cells), injected intravenously 18-24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF-ß expression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays. Conclusions. This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.

Peripheral post-ischemic vascular repair is impaired in a murine model of Alzheimer's disease.

The pathophysiology of sporadic Alzheimer's disease (AD) remains uncertain. Along with brain amyloid-ß (Aß) deposits and neurofibrillary tangles, cerebrovascular dysfunction is increasingly recognized as fundamental to the pathogenesis of AD. Using an experimental model of limb ischemia in transgenic APPPS1 mice, a model of AD (AD mice), we showed that microvascular impairment also extends to the peripheral vasculature in AD. At D70 following femoral ligation, we evidenced a significant decrease in cutaneous blood flow (- 29%, P < 0.001), collateral recruitment (- 24%, P < 0.001), capillary density (- 22%; P < 0.01) and arteriole density (- 28%; P < 0.05) in hind limbs of AD mice compared to control WT littermates. The reactivity of large arteries was not affected in AD mice, as confirmed by unaltered size, and vasoactive responses to pharmacological stimuli of the femoral artery. We identified blood as the only source of Aß in the hind limb; thus, circulating Aß is likely responsible for the impairment of peripheral vasculature repair mechanisms. The levels of the majority of pro-angiogenic mediators were not significantly modified in AD mice compared to WT mice, except for TGF-ß1 and PlGF-2, both of which are involved in vessel stabilization and decreased in AD mice (P = 0.025 and 0.019, respectively). Importantly, endothelin-1 levels were significantly increased, while those of nitric oxide were decreased in the hind limb of AD mice (P < 0.05). Our results suggest that vascular dysfunction is a systemic disorder in AD mice. Assessment of peripheral vascular function may therefore provide additional tools for early diagnosis and management of AD.

Male sex may be associated with higher metabolic risk in first-episode schizophrenia patients: A preliminary study.

BACKGROUND: High incidence of metabolic syndrome has been evidenced in schizophrenia patients. However, gender-specific relationship with risk of metabolic disorders in first-episode schizophrenia has received poor systematic study. AIM: We aimed at exploring the impact of sex difference on the parameters of glucolipid metabolism in first-episode psychosis schizophrenia (FEP) patients. METHODS: We performed a post hoc analysis of data from our previously performed clinical trial. A total of 60 FEP patients and 28 healthy sex- and age-matched volunteers were included. Blood glucose and lipid metabolic profiles, as well as schizophrenia-related clinical symptoms were assessed. The body mass index, level of blood insulin and the homeostasis model assessment-insulin resistance index (HOMA-IR) were measured. RESULTS: The FEP patients demonstrated significant increases in blood insulin concentration, insulin resistance and blood triglyceride when compared with healthy controls. In FEP patients, there were no differences in psychopathology measurements between the genders. BMI and HOMA-IR were significantly greater in male vs female FEP patients. In addition, a more severe dyslipidemia was also observed in male FEP patients, including an increased triglyceride level, an augmented LDL content and a decreased HDL concentration. Multivariate linear regression analysis demonstrated that the gender was significantly correlated to HOMA-IR. CONCLUSION: These preliminary results suggest that male FEP patients may be more predisposed to insulin resistance and dyslipidemia than female FEP patients. These results could contribute to the understanding of prevention and treatment of metabolic syndrome in FEP patients.

Antitumoral Effect of Mural Cells Assessed With High-Resolution MRI and Fluorescence Microscopy.

OBJECTIVE: The purpose of this study was to detect labeled mural cells in vivo and study their therapeutic effect on tumor growth and on functional changes in the vascular network by use of MRI and fibered confocal fluorescence microscopy (FCFM). MATERIALS AND METHODS: Twenty-eight mice were allocated to the following three groups 7 days after injection of TC1 tumor cells (C157 black 6): control, no injection (n = 7); sham, injection of phosphate-buffered saline solution (n = 10); and treated, injection of human mural cells (n = 11). Tumor growth was measured with calipers. Labeled mural cells were tracked with high-resolution MRI and FCFM. Microvessel density was assessed with MRI and FCFM, and the findings were compared with the histologic results. RESULTS: Tumor growth was significantly slowed in the treated group starting on day 10 (p = 0.001). Round signal-intensity voids were observed in the center of six of seven tumors treated with magnetically labeled mural cells. Positive staining for iron was observed in histologic sections of two of five of these tumors. Microvessel density measured with FCFM was greater in the treated mice (p = 0.03). Flow cytometry revealed viable human mural cells only in treated tumors. CONCLUSION: In this study, imaging techniques such as high-resolution MRI and FCFM showed the therapeutic effect of mural cell injection on tumor growth and microvessel function.

Characterization of nerve and microvessel damage and recovery in type 1 diabetic mice after permanent femoral artery ligation.

Neuropathy is the most common complication of the peripheral nervous system during the progression of diabetes. The pathophysiology is unclear but may involve microangiopathy, reduced endoneurial blood flow, and tissue ischemia. We used a mouse model of type 1 diabetes to study parallel alterations of nerves and microvessels following tissue ischemia. We designed an easily reproducible model of ischemic neuropathy induced by irreversible ligation of the femoral artery. We studied the evolution of behavioral function, epineurial and endoneurial vessel impairment, and large nerve myelinated fiber as well as small cutaneous unmyelinated fiber impairment for 1 month following the onset of ischemia. We observed a more severe hindlimb dysfunction and delayed recovery in diabetic animals. This was associated with reduced density of large arteries in the hindlimb and reduced sciatic nerve epineurial blood flow. A reduction in sciatic nerve endoneurial capillary density was also observed, associated with a reduction in small unmyelinated epidermal fiber number and large myelinated sciatic nerve fiber dysfunction. Moreover, vascular recovery was delayed, and nerve dysfunction was still present in diabetic animals at day 28. This easily reproducible model provides clear insight into the evolution over time of the impact of ischemia on nerve and microvessel homeostasis in the setting of diabetes. © 2015 Wiley Periodicals, Inc.

Hypertension accelerates the progression of Alzheimer-like pathology in a mouse model of the disease.

Cerebrovascular impairment is frequent in patients with Alzheimer disease and is believed to influence clinical manifestation and severity of the disease. Cardiovascular risk factors, especially hypertension, have been associated with higher risk of developing Alzheimer disease. To investigate the mechanisms underlying the hypertension, Alzheimer disease cross talk, we established a mouse model of dual pathology by infusing hypertensive doses of angiotensin II into transgenic APPPS1 mice overexpressing mutated human amyloid precursor and presenilin 1 proteins. At 4.5 months, at the early stage of disease progression, only hypertensive APPPS1 mice presented impairment of temporal order memory performance in the episodic-like memory task. This cognitive deficit was associated with an increased number of cortical amyloid deposits (223±5 versus 207±5 plaques/mm(2); P<0.05) and a 2-fold increase in soluble amyloid levels in the brain and in plasma. Hypertensive APPPS1 mice presented several cerebrovascular alterations, including a 25% reduction in cerebral microvessel density and a 30% to 40% increase in cerebral vascular amyloid deposits, as well as a decrease in vascular endothelial growth factor A expression in the brain, compared with normotensive APPPS1 mice. Moreover, the brain levels of nitric oxide synthase 1 and 3 and the nitrite/nitrate levels were reduced in hypertensive APPPS1 mice (by 49%, 34%, and 33%, respectively, compared with wild-type mice; P<0.05). Our results indicate that hypertension accelerates the development of Alzheimer disease-related structural and functional alterations, partially through cerebral vasculature impairment and reduced nitric oxide production.

Netrin-1 controls sympathetic arterial innervation.

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Orthotopic animal model of pseudomyxoma peritonei: An in vivo model to test anti-angiogenic drug effects.

Pseudomyxoma peritonei (PMP) is an uncommon peritoneal mucinous carcinomatosis confined to the peritoneal cavity. The rarity of PMP in humans makes evaluation of the disease biological features and new therapeutic strategies difficult. Accordingly, there is a need for animal models of PMP. Human PMP tissue was i.p. grafted and grown into nude mice, then constituted into reliable and reproducible orthotopic models. Histological and immunostaining analysis was performed. Bevacizumab was injected twice a week either during tumor growth or after cytoreductive surgery. In vivo imaging of tumor angiogenesis was performed using barium sulfate or isolectin microangiography and Doppler ultrasonography of the superior mesenteric artery. Tumor angiogenesis was confirmed by the presence of tortuous vascular networks with high levels of expression of CD31, vascular endothelial cadherin, and desmin. Doppler ultrasonography of the superior mesenteric artery revealed a twofold increase in blood flow velocity compared with tumor-free mice (P < 0.001). Bevacizumab administration was correlated with the normalization of tumor vascularity when injected during tumor growth and with the stabilization of the histological and hemodynamic findings when injected after cytoreductive surgery. Our PMP models mimic human PMP. Our results confirmed the presence of tumor angiogenesis related to PMP growth. Our murine model allows researchers to actually bench test and evaluate, in preclinical studies, the efficacy of new therapeutic strategies and anti-angiogenic therapies.

Relationship between insulin resistance, dyslipidaemia and positive symptom in Chinese antipsychotic-naive first-episode patients with schizophrenia.

Controversial results concerning insulin resistance and lipid metabolism have been reported in antipsychotic-naive first-episode psychosis (AN-FEP) patients with schizophrenia of different countries. We aimed at determining whether schizophrenia-related psychopathology was associated with insulin resistance and/or dyslipidaemia in Chinese patients with AN-FEP schizophrenia. A cross-sectional study was performed in Chinese patients newly diagnosed with schizophrenia (n = 49, antipsychotic-naïve or antipsychotic medications< 2 weeks) and healthy volunteers (n = 30). The serum levels of insulin and triglyceride levels as well as homeostasis model of assessment-insulin resistance (HOMA-IR) index were significantly increased in AN-FEP patients, when compared with healthy volunteers. The gender difference had a significant impact on the insulin resistance and dyslipidaemia in these AN-FEP subjects. Multiple linear regression analysis demonstrated that the severity of positive symptoms of schizophrenia was negatively related to insulin resistance. No difference of serum glucose level, total cholesterol content, body mass index (BMI) and smoking status was detected between patients with schizophrenia and healthy controls. In conclusion, Chinese AN-FEP patients were more prone to insulin resistance and dyslipidaemia as compared to the healthy population, which is negatively correlated to positive symptoms. The results may contribute to the understanding of the relationship between the glucose/lipidaemia metabolic dysfunction and the psychopathology in patients with schizophrenia.

Hepatic ischemia-reperfusion increases circulating bone marrow-derived progenitor cells and tumor growth in a mouse model of colorectal liver metastases.

BACKGROUND: Hepatic pedicle clamping is often required to reduce blood loss and transfusion during liver resection. However, the question remains whether use of hepatic pedicle clamping promotes tumor growth. Endothelial progenitor cells (EPCs) are mobilized from bone marrow in response to tissue ischemia, which allows neovascularization of ischemic tissue. It has been suggested that EPCs are involved in tumor progression. We hypothesized that hepatic ischemia reperfusion (I/R)-induced mobilization of EPCs could enhance growth of microscopic tumor, therefore promoting liver metastasis in a mouse model of colorectal cancer. MATERIALS AND METHODS: We used mouse models of hepatic I/R and hind limb ischemia. For comparison, we studied mice that underwent limb ischemia as positive controls of EPC mobilization. At day 0, we divided 40 mice into four groups: hepatic I/R, hind limb ischemia, combined hepatic I/R and hind limb ischemia, and control (sham midline incision laparotomy). At day 2, we induced liver metastasis in all mice by injecting CT-26 cells into the spleen. Time-dependent circulating EPCs were determined by flow cytometry. We evaluated liver metastasis and microvascular density on day 21. RESULTS: The number of circulating progenitor cells increased rapidly in the ischemic groups compared with the control group. Hepatic I/R significantly increased tumor outgrowth compared with the control group. Increased tumor growth was associated with enhanced CD31-positive microvascular density in liver tissue. CONCLUSIONS: Hepatic I/R leads to mobilization of bone marrow-derived EPCs and enhanced intra-hepatic angiogenesis, which is associated with increased tumor burden in an animal model of colorectal liver metastasis.

Ephrin-B2-activated peripheral blood mononuclear cells from diabetic patients restore diabetes-induced impairment of postischemic neovascularization.

We hypothesized that in vitro treatment of peripheral blood mononuclear cells (PB-MNCs) from diabetic patients with ephrin-B2/Fc (EFNB2) improves their proangiogenic therapeutic potential in diabetic ischemic experimental models. Diabetes was induced in nude athymic mice by streptozotocin injections. At 9 weeks after hyperglycemia, 10(5) PB-MNCs from diabetic patients, pretreated by EFNB2, were intravenously injected in diabetic mice with hindlimb ischemia. Two weeks later, the postischemic neovascularization was evaluated. The mechanisms involved were investigated by flow cytometry analysis and in vitro cell biological assays. Paw skin blood flow, angiographic score, and capillary density were significantly increased in ischemic leg of diabetic mice receiving EFNB2-activated diabetic PB-MNCs versus those receiving nontreated diabetic PB-MNCs. EFNB2 bound to PB-MNCs and increased the adhesion and transmigration of PB-MNCs. Finally, EFNB2-activated PB-MNCs raised the number of circulating vascular progenitor cells in diabetic nude mice and increased the ability of endogenous bone marrow MNCs to differentiate into cells with endothelial phenotype and enhanced their proangiogenic potential. Therefore, EFNB2 treatment of PB-MNCs abrogates the diabetes-induced stem/progenitor cell dysfunction and opens a new avenue for the clinical development of an innovative and accessible strategy in diabetic patients with critical ischemic diseases.

Towards the therapeutic use of vascular smooth muscle progenitor cells.

Recent advances in the development of alternative proangiogenic and revascularization processes, including recombinant protein delivery, gene therapy, and cell therapy, hold the promise of greater efficacy in the management of cardiovascular disease in the coming years. In particular, vascular progenitor cell-based strategies have emerged as an efficient treatment approach to promote vessel formation and repair and to improve tissue perfusion. During the past decade, considerable progress has been achieved in understanding therapeutic properties of endothelial progenitor cells, while the therapeutic potential of vascular smooth muscle progenitor cells (SMPC) has only recently been explored; the number of the circulating SMPC being correlated with cardiovascular health. Several endogenous SMPC populations with varying phenotypes have been identified and characterized in the peripheral blood, bone marrow, and vascular wall. While the phenotypic entity of vascular SMPC is not fully defined and remains an evolving area of research, SMPC are increasingly recognized to play a special role in cardiovascular biology. In this review, we describe the current approaches used to define vascular SMPC. We further summarize the data on phenotype and functional properties of SMPC from various sources in adults. Finally, we discuss the role of SMPC in cardiovascular disease, including the contribution of SMPC to intimal proliferation, angiogenesis, and atherosclerotic plaque instability as well as the benefits resulting from the therapeutic use of SMPC.

Netrin-4 delays colorectal cancer carcinomatosis by inhibiting tumor angiogenesis.

A close relationship between tumor angiogenesis, growth, and carcinomatosis has been observed. Netrin-4 (NT-4) has been shown to regulate angiogenic responses. We aimed to examine the effects of NT-4 on colon tumor angiogenesis, growth, and carcinomatosis. We showed that NT-4 was expressed in human colon cancer cells (LS174). A 20-fold increase in NT-4 expression was stably induced by NT-4 pcDNA in LS174 cells. In vivo, a Matrigel angiogenesis assay showed that NT-4 overexpression altered vascular endothelial growth factor (VEGF)/basic fibroblast growth factor-induced angiogenesis. In nude mice with LS174 xenografts, NT-4 overexpression inhibited tumor angiogenesis and growth. In addition, these NT-4-involved inhibitory effects were associated with decreased tumor cell proliferation and increased tumor cell apoptosis. Using an orthotopic peritoneal carcinomatosis model, we demonstrated that NT-4 overexpression decreased colorectal cancer carcinomatosis. Moreover, carcinomatosis-related ascites formation was significantly decreased in mice transplanted with NT-4 LS174 cells versus control LS174 cells. The antiangiogenic activity of NT-4 was probably mediated by binding to its receptor neogenin. Netrin-4 had a direct effect on neither in vitro apoptosis and proliferation of cultured LS174 cells nor the VEGF-induced acute increase in vascular permeability in vivo. We propose that NT-4 overexpression decreases tumor growth and carcinomatosis, probably via an antiangiogenic effect, underlying the potential therapeutic interest in NT-4 in the treatment of colorectal cancer growth and carcinomatosis.