Netrin-1 Treatment Research Articles

Pharmacological postconditioning treatment of myocardial infarction with Netrin-1

The present study investigated whether pharmacological postconditoning with netrin-1 is cardioprotective against ischemia reperfusion (I/R) injury, and the underlying signaling mechanisms. Langendorff perfused hearts isolated from wild-type (WT) C57BL/6 or DCC+/- mice underwent a 20-min of ischemia, followed by a 60-min of reperfusion, in the presence or absence of netrin-1, or netrin-1 in combination with U0126 (MEK1/2 inhibitor), or PTIO (nitric oxide/NO scavenger). In WT mice, netrin-1 postconditioning dramatically reduced infarct size to 17.0±2.5%, from 40.5±4.2% in the untreated I/R group. U0126 or PTIO alone had no effect on infarct size but abolished the effects of netrin-1. The protective effect of netrin-1 was markedly diminished in DCC+/- mice (44.5±2% vs. 15±2.6 % for infract size in DCC+/- vs. DCC+/+ group). Our results indicate that netrin-1, given as a pharmacological postconditioning agent, induces cardioprotection via a DCC-dependent mechanism that involves ERK1/2 activation and NO production. Combined with our previous findings, netrin-1 treatment proves to be extremely and consistently beneficial whenever delivered to the heart, establishing its substantial promises for being developed into a robust therapeutic strategy for acute myocardial infarction.

Netrin-1 Attenuates Cardiac Ischemia Reperfusion Injury and Generates Alternatively Activated Macrophages

Ischemia reperfusion (IR) injury is a major issue in cardiac transplantation and inflammatory processes play a major role in myocardial IR injury. Netrin-1 is a laminin-related protein identified as a neuronal guidance cue and netrin-1 expressed outside the nervous system inhibits migration of leukocytes in vitro and in vivo and attenuates inflammation-mediated tissue injury. In our study, hearts of C57BL/6 mice were flushed and stored in cold Bretschneider solution for 8 h and then transplanted into syngeneic recipient. We found that netrin-1 decreased cardiomyocyte apoptosis and recruitment of neutrophils and macrophages. Troponin T (TnT) production on 24 h after myocardial IR injury was reduced by netrin-1 administration. Cardiac output at 60 mmHg of afterload pressure was significantly increased in hearts with netrin-1 administration (IR + Netrin-1: 59.9 ± 5.78 ml/min; IR: 26.2 ± 4.3 ml/min; P < 0.05). Netrin-1 treatment increased expression of the alternatively activated macrophage (AAM) markers arginase-1 (Arg-1) and mannose receptor (MR) and promoted proliferator-activated receptor γ (PPARγ) expression in cardiac allograft. Furthermore, decreased TnT expression and reduced allograft infiltration of neutrophils and monocytes/macrophages by netrin-1 was abolished with addition of PPARγ antagonist. In conclusion, netrin-1 attenuates cardiac IR injury and generates AAM which contributes to the protective effect of netrin-1.

Protective role for netrin-1 during diabetic nephropathy

Recent studies implicate neuronal guidance molecules in the orchestration of inflammatory events. For example, previous studies demonstrate a functional role for netrin-1 in attenuating acute kidney injury. Here, we hypothesized a kidney-protective role for netrin-1 during chronic kidney disease, such as occurs during diabetic nephropathy. To study the role of netrin-1 during diabetic nephropathy, we induced diabetes in mice at the age of 8 weeks by streptocotozin (STZ) treatment. Sixteen weeks after STZ treatment, we examined the kidneys. Initial studies in wild-type mice demonstrated robust induction of renal, urinary, and plasma netrin-1 protein levels during diabetic nephropathy. Subsequent genetic studies in mice with partial netrin-1 deficiency (Ntrn1(+/-) mice) revealed a more severe degree of diabetic nephropathy, including more severe loss of kidney function (albuminuria, glomerular filtration rate, histology). We subsequently performed pharmacologic studies with recombinant netrin-1 treatment given continuously via osmotic pump. Indeed, netrin-1 treatment was associated with attenuated albuminuria and improved histologic scores for diabetic nephropathy compared to controls. Consistent with previous studies implicating purinergic signaling in netrin-1-elicited tissue protection, mice deficient in the Adora2b adenosine receptor were not protected. Taken together, these studies demonstrate a functional role for endogenous netrin-1 in attenuating diabetic kidney disease.

Netrin-1 Attenuates the Progression of Renal Dysfunction by Inhibiting Peritubular Capillary Loss and Hypoxia in 5/6 Nephrectomized Rats

Background/Aims: The aim of this study was to investigate the effect of netrin-1 on peritubular capillary (PTC) loss and hypoxia in 5/6 nephrectomized (Nx) rats. Methods: Male Sprague-Dawley rats were divided into three groups (n = 10 rats/group): sham-operated rats treated with control adenovirus; 5/6 Nx rats treated with control adenovirus; and 5/6 Nx rats treated with recombinant adenovirus mediated netrin-1 gene (Ad-netrin-1) therapy. Rats were killed 12 weeks after surgery. Blood urea nitrogen (BUN), serum creatinine (Scr) and 24-h urinary albumin excretion rates were measured. Pathological changes in renal tissues were analyzed histologically. The concentration of netrin-1, CD34, and hypoxia-inducible factor-1α (HIF-1α) were analyzed by immunohistochemistry, Western blotting and real-time PCR. Results: Renal function and histopathological damage were significantly improved in Adnetrin-1 treated 5/6 Nx rats, compared with rats treated with the control adenovirus in the 5/6 Nx group. Furthermore, Ad-netrin-1 treatment induced a significant increase in renal PTC density, accompanied by a significant decrease in HIF-1α expression. Conclusion: Adenovirus mediated netrin-1 treatment attenuates PTC damage, relieves tissues hypoxia and improves renal function, thus alleviating renal pathological changes and interstitial fibrosis in 5/6 Nx rats.

Abstract 17337: Postconditioning with Netrin-1 Protects Against Myocardial Ischemia-Reperfusion Injury Via a DCC/ERK1/2/eNOS Mechanism

Given as a pre-conditioning mimetic, we have previously shown that netrin-1 markedly reduces myocardial ischemia-reperfusion (I/R) injury by increasing nitric oxyde (NO) bioavailability. However, its potential beneficial effect remains unknown if given as a post-conditioning agent. Langendorff perfused hearts isolated from C57BL/6 wild-type (WT) mice or DCC+/- mice underwent 20-min of global ischemia followed by 60-min of reperfusion (n=6). DCC (deleted in colorectal cancer) is one of the netrin-1 receptors we found to be however specifically involved in netrin-1 pre-conditioning induced cardioprotection. Netrin-1 was infused post-conditionally at the onset of reperfusion at 100 ng/mL concentration with and without UO126, a specific MEK1/2 inhibitor or 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a NO-scavenger. In WT mice, netrin-1 postconditioning significantly reduced infarct size to 17±2.5% from 43.3±4.6% in the untreated control I/R group. This phenomenon is now further confirmed using an in vivo model of myocardial ischemic injury, 30-min coronary occlusion followed by 24-hr reperfusion. UO126 or PTIO administration alone had no effect on infarct size (respectively 44.8±3% and 39.8±1.7% vs 43.3±4.6% in the untreated control I/R group) but abolished the effects of netrin-1, implicating intermediate roles of MEK/ERK1/2 and NO (respectively 44.8±2% and 43.5±2.9% vs 17±2.5% in the netrin-1 treated I/R group). Indeed, ERK1/2 and eNOS phosphorylation was significantly upregulated by netrin-1 treatment. Interestingly, the protective effect of netrin-1 was markedly diminished in the DCC+/- mice (infarct size increased to 44.5±2% from 15±2.6 % in the DCC+/+ group), indicating a dependency on DCC of netrin-1 post-conditoning induced cardioprotection. Netrin-1 also feed-forwardly upregulated DCC protein expression. Taken together, these results strongly suggest that netrin-1 protects against I/R injury in the murine heart as a post-conditioning mimetic by increasing NO production via activation of a ERK1/2/eNOS pathway, which also involves DCC activation and upregulation. These findings demonstrate that netrin-1 can serve as an innovative and powerful agent for acute treatment of myocardial infarction.

Neuronal Guidance Protein Netrin-1 Induces Differentiation in Human Embryonal Carcinoma Cells

Pluripotent cells within embryonal carcinoma (EC) can differentiate in vivo or in vitro on treatment with specific agents. Differentiating EC cells express lower levels of stem cell-related genes, such as Cripto-1. We show that migration of human EC cells (NTERA/2 and NCCIT) can be reduced following treatment with the guidance molecule Netrin-1. Moreover, Netrin-1 treatment increased the levels of beta-III tubulin, glial filament acidic protein, Nestin, and gamma-aminobutyric acid and reduced the expressions of Cripto-1, Nanog, and Oct4 in EC cells. These Netrin-1-induced effects in the EC cells were mediated via binding of Netrin-1 to the Neogenin receptor and activation of SHP-2, resulting in increased levels of inactive phosphorylated c-src((Y527)). These results suggest that Netrin-1 can induce neuroectodermal-like differentiation of human EC cells by affecting c-src signaling via SHP-2 activation and regulation of Nanog, Oct4, and Cripto-1 expressions.

NETRIN-1 SIGNALING AND GnRH NEURONAL MIGRATION

Recent evidence suggests a role for netrin-1 in regulation of GnRH neuronal migration. The purpose of the current study was to elucidate the downstream signaling pathway utilized by netrin-1 in GnRH neurons. Toward this end, we examined the co-localization and netrin-1 regulation of the netrin receptor, deleted in colorectal cancer (DCC), and its downstream binding partners, focal adhesion kinase (FAK) and myosin- X (Myo-X) in GnRH neurons. Both “migratory” (NLT, Gn11) and “postmigratory” (GT1-7) GnRH neuronal cell lines were utilized in the studies on GnRH neuronal migration and co-localization. Additionally, in vivo studies were performed to examine for co-localization of netrin-1 downstream signaling partners in migrating GnRH neurons in vivo from embryonic day 12–16 (E12–E16) in the mouse. The results of the study revealed that netrin-1, DCC, FAK and Myo-X are colocalized in subsets of GnRH neurons in vitro and in vivo. Furthermore, netrin-1 treatment in vitro stimulated GnRH neuronal migration and induced reorganization of actin cytoskeleton and focal adhesions in GnRH neuronal cell lines. The studies suggest that netrin-1 action on GnRH neuronal migration may involve netrin-1 mediated signaling via DCC-FAK and/or DCC-Myo-X. (This work was supported by a grant from the Medical College of Georgia Intramural Grant IRP program) (poster)