Differentiation of neural stem cells into tyrosine hydroxylase (TH)-expressing cells was studied by cell transplantation into the various brain regions of rats that had received 6-hydroxydopamine ...lesion of the nigrostriatal pathway. Approximately 13.6–16.1% of survived precursor cells acquired neuronal-like features by expressing the neuronal marker doublecortin. Similarly, 20.7–25.7% of survived precursor cells differentiated into astrocytes following transplantation. Immunohistochemical analysis revealed that a fraction of the grafted precursor cells in the anterior part of the medial forebrain bundle (MFB), but not in the striatum and the substantia nigra of the lesioned side, showed strong TH immunoreactivity. This suggests that MFB is more permissive for induction of TH to the striatal precursor cells in vivo. The results further exemplify the potential of neural stem cells and the property of site-specific differentiation when the cells were transplanted to the dopaminergic system of the adult brain.
Vascular endothelial growth factor B (VEGF-B) was discovered a long time ago. However, its role in hyperglycemia- and VEGF-A inhibition-induced retinal apoptosis remains unknown thus far. Yet, drugs ...that can block VEGF-B are being used to treat patients with diabetic retinopathy and other ocular neovascular diseases. It is therefore urgent to have a better understanding of the function of VEGF-B in these pathologies. Here, we report that both streptozotocin (STZ)-induced diabetes in rats and Macugen intravitreal injection in mice leads to retinal apoptosis in retinal ganglion cell and outer nuclear layers respectively. Importantly, VEGF-B treatment by intravitreal injection markedly reduced retinal apoptosis in both models. We further reveal that VEGF-B and its receptors, vascular endothelial growth factor 1 (VEGFR1) and neuropilin 1 (NP1), are abundantly expressed in rat retinae and choroids and are upregulated by high glucose with concomitant activation of Akt and Erk. These data highlight an important function of VEGF-B in protecting retinal cells from apoptosis induced by hyperglycemia and VEGF-A inhibition. VEGF-B may therefore have a therapeutic potential in treating various retinal degenerative diseases, and modulation of VEGF-B activity in the eye needs careful consideration.
Platelet-derived growth factor (PDGF)-C is a member of the PDGF family and is critical for neuronal survival in the central nervous system. We studied the possible survival and antiapoptotic effects ...of PDGF-C on focal retinal lesions in Ccl2−/−/Cx3cr1−/− on C57BL/6N Crb1rd8 (DKO rd8) background mice, a model for progressive and focal retinal degeneration. We found no difference in transcript and protein expression of PDGF-C in the retina between DKO rd8 mice and wild type (WT, C57BL/6N). Recombinant PDGF-CC protein (500 ng/eye) was injected intravitreally into the right eye of DKO rd8 mice with phosphate-buffered saline as controls into the left eye. The retinal effects of PDGF-C were assessed by fundoscopy, ocular histopathology, A2E levels, apoptotic molecule analysis, and direct flat mount retinal vascular labeling. We found that the PDGF-CC-treated eyes showed slower progression or attenuation of the focal retinal lesions, lesser photoreceptor and retinal pigment epithelial degeneration resulting in better-preserved photoreceptor structure. Lower expression of apoptotic molecules was detected in the PDGF-CC-treated eyes than in controls. In addition, no retinal neovascularization was observed after PDGF-CC treatment. Our results demonstrate that PDGF-C potently ameliorates photoreceptor degeneration via the suppression of apoptotic pathways without inducing retinal angiogenesis. The protective effects of PDGF-C suggest a novel alternative approach for potential age-related retinal degeneration treatment.
β1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide ...exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on β1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2-mediated αVβ3-integrin-recycling and β1-integrin endocytosis and specifically of the active/matrix-bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.
Platelet-derived growth factor-DD (PDGF-DD) is a recently discovered member of the PDGF family. The role of PDGF-DD in pathological angiogenesis and the underlying cellular and molecular mechanisms ...remain largely unexplored. In this study, using different animal models, we showed that PDGF-DD expression was up-regulated during pathological angiogenesis, and inhibition of PDGF-DD suppressed both choroidal and retinal neovascularization. We also demonstrated a novel mechanism mediating the function of PDGF-DD. PDGF-DD induced glycogen synthase kinase-3β (GSK3β) Ser9 phosphorylation and Tyr216 dephosphorylation in vitro and in vivo, leading to increased cell survival. Consistently, GSK3β activity was required for the antiangiogenic effect of PDGF-DD targeting. Moreover, PDGF-DD regulated the expression of GSK3β and many other genes important for angiogenesis and apoptosis. Thus, we identified PDGF-DD as an important target gene for antiangiogenic therapy due to its pleiotropic effects on vascular and non-vascular cells. PDGF-DD inhibition may offer new therapeutic options to treat neovascular diseases.
Anti-VEGF-A therapy has proven to be effective for many neovascular diseases. However, drug resistance to anti-VEGF-A treatment can develop. Also, not all patients with neovascular diseases are ...responsive to anti-VEGF-A treatment. The mechanisms underlying these important issues remain unclear. In this study, using different model systems, we found that inhibition of VEGF-A directly upregulated PDGF-CC and its receptors in multiple cell types in pathological angiogenesis in vitro and in vivo. Importantly, we further revealed that combinatorial targeting of VEGF-A and PDGF-CC suppressed pathological angiogenesis more efficiently than monotherapy. Given the potent angiogenic activity of PDGF-CC, our findings suggest that the development of resistance to anti-VEGF-A treatment may be caused by the compensatory upregulation of PDGF-CC, and combined inhibition of VEGF-A and PDGF-CC may have therapeutic advantages in treating neovascular diseases.
Abstract only β1-integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a ...guanine nucleotide exchange factor (GEF) for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated silencing of Brag2 significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and migration of HUVEC (49 % inhibition). Transfection with Brag2-siRNA differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and EC adhesion on β1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin-ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2 mediated recycling of αVβ3-integrins and endocytosis of β1-integrins and specifically of the active/matrix bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin-endocytosis. Furthermore, we found that Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin-endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Moreover, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.
A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered (1). This uniqueness has made the ...cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect (2,3). Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors (4), it is used here to induce angiogenesis in the cornea.
VEGF is believed to be a master regulator in both developmental and pathological angiogenesis. The role of PDGF-C in angiogenesis, however, is only at the beginning of being revealed. We and others ...have shown that PDGF-C is a critical player in pathological angiogenesis because of its pleiotropic effects on multiple cellular targets. The angiogenic pathways induced by PDGF-C are, to a large extent, VEGF-independent. These pathways may include, but not limited to, the direct effect of PDGF-C on vascular cells, the effect of PDGF-C on tissue stroma fibroblasts, and its effect on macrophages. Taken together, the pleiotropic, versatile and VEGF-independent angiogenic nature of PDGF-C has placed it among the most important target genes for antiangiogenic therapy.
To begin to identify novel protein(s) that acts on nigral dopaminergic (DA) neurons, we characterized trophic effects of DA-depleted striatum on survival of fetal DA neurons in the present study. ...Treatment of ventral mesencephalic cultures with the striatal extracts delayed DA cell death in a dose-dependent manner. This effect was partially dependent on brain-derived neurotrophic factor (BDNF), but not glial cell line-derived neurotrophic factor (GDNF), present in the extracts. Furthermore, we addressed the hypothesis that the striatum-derived substances can elicit DA phenotypic expression of striatal cells in cultures. The striatal extract was found to be able to induce expression of tyrosine hydroxylase in cultured striatal cells in the presence of dopamine. These data suggest that denervation of the striatum resulted in production of neurotrophic factors, including BDNF and as-yet-unidentified trophic substances, which may be responsible for the increased survival and DA phenotype expression in DA neurons.
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