Regulated Cell Death of Retinal Ganglion Cells in Glaucoma: Molecular Insights and Therapeutic Potentials.

Glaucoma is a group of diseases characterized by the degeneration of retinal ganglion cells (RGCs) and progressive, irreversible vision loss. High intraocular pressure (IOP) heightens the likelihood of glaucoma and correlates with RGC loss. While the current glaucoma therapy prioritizes lower the IOP; however, RGC, and visual loss may persist even when the IOP is well-controlled. As such, discovering and creating IOP-independent neuroprotective strategies for safeguard RGCs is crucial for glaucoma management. Investigating and clarifying the mechanism behind RGC death to counteract its effects is a promising direction for glaucoma control. Empirical studies of glaucoma reveal the role of multiple regulated cell death (RCD) pathways in RGC death. This review delineates the RCD of RGCs following IOP elevation and optic nerve damage and discusses the substantial benefits of mitigating RCD in RGCs in preserving visual function.

Chronic lead exposure reduces doublecortin-expressing immature neurons in young adult guinea pig cerebral cortex.

BACKGROUND: Chronic lead (Pb) poisoning remains an environmental risk especially for the pediatric population, and it may affect brain development. Immature neurons expressing doublecortin (DCX+) exist around cortical layer II in various mammals, including adult guinea pigs and humans. Using young adult guinea pigs as an experimental model, the present study explored if chronic Pb exposure affects cortical DCX + immature neurons and those around the subventricular and subgranular zones (SVZ, SGZ). RESULTS: Two month-old guinea pigs were treated with 0.2% lead acetate in drinking water for 2, 4 and 6 months. Blood Pb levels in these animals reached 10.27 ± 0.62, 16.25 ± 0.78 and 19.03 ± 0.86 µg/dL at the above time points, respectively, relative to ~3 µg/dL in vehicle controls. The density of DCX + neurons was significantly reduced around cortical layer II, SVZ and SGZ in Pb-treated animals surviving 4 and 6 months relative to controls. Bromodeoxyuridine (BrdU) pulse-chasing studies failed to find cellular colocalization of this DNA synthesis indicator in DCX + cells around layer II in Pb-treated and control animals. These cortical immature neurons were not found to coexist with active caspase-3 or Fluoro-Jade C labeling. CONCLUSION: Chronic Pb exposure can lead to significant reduction in the number of the immature neurons around cortical layer II and in the conventional neurogenic sites in young adult guinea pigs. No direct evidence could be identified to link the reduced cortical DCX expression with alteration in local neurogenesis or neuronal death.

Timosaponin-BII inhibits the up-regulation of BACE1 induced by ferric chloride in rat retina.

BACKGROUND: Our previous studies indicated that oxidative stress up-regulated the expression of ß-amyloid precursor protein cleavage enzyme-1 (BACE1) in rat retina. Pharmacological reports have shown Timosaponin-BII, a purified extract originating from Chinese medical herb Rhizoma Anemarrhenae, is characterized as an antioxidant. Our present study aimed to determine whether Timosaponin-BII affected the expression of BACE1, ß-amyloid precursor protein cleavage production of Aß1-40 and ß-C-terminal fragment (ß-CTF) in rat retina, which were pre-treated with the oxidizing agent (solution of FeCl3). RESULTS: Few distinctions of BACE1 distribution were observed among all groups (normal control group, model group, Timosaponin-BII treated and vehicle control groups). Rat retinas in model group and vehicle control group manifested an apparent up-regulation of BACE1 expression. Meanwhile, the level of malonaldehyde (MDA), Aß1-40 and ß-CTF were increased. However, when comparing with the vehicle control group, the retinas in Timosaponin-BII treated group showed significantly less BACE1 (p<0.05) and accumulated less Aß1-40 or ß-CTF (p<0.05). It also showed significantly decreased level of MDA (p<0.05) and prolonged partial thromboplastin time (p<0.05). CONCLUSION: Our data suggested that Timosaponin-BII remarkably inhibited the up-regulation of BACE1 and reduced the over-production of ß-CTF and Aß in rat retina, which was induced by FeCl3. The mechanism of Timosaponin-BII on BACE1 expression may be related to its antioxidant property.

Angiotensin II-stimulated collagen synthesis in aortic adventitial fibroblasts is mediated by connective tissue growth factor.

Angiotensin II (Ang II), a potent mediator of vascular remodeling, can stimulate the synthesis of extracellular matrix in vascular cells. Recent studies indicate that connective tissue growth factor (CTGF) is involved in collagen synthesis. There is also increasing evidence that adventitial fibroblasts (AFs) are actively involved in vascular remodeling. However, whether collagen synthesis by AFs is mediated by CTGF, or whether it is relevant to Ang II, has not been studied. The present study was conducted to determine whether CTGF is expressed in AFs, and if so, whether the CTGF produced by AFs participates in collagen synthesis. The AFs were isolated from thoracic aorta of Wistar-Kyoto rats (WKY). The expression of CTGF was measured by Western blot or real-time PCR. Collagen synthesis was assessed by [(3)H]proline incorporation. Our results suggested that CTGF was expressed in AFs and secreted into medium. Ang II increased CTGF mRNA and protein expression in a time- and dose-dependent manner, with the maximal protein increase occurring at 24 h with an Ang II dose of 10(-7) mol/L, and this increase was inhibited by the Ang II receptor type 1 (AT(1)-R) antagonist losartan, but not by the Ang II receptor type 2 (AT(2)-R) antagonist PD123319. Ang II dose-dependently stimulated the incorporation of [(3)H]proline into cultured AFs, and this effect was inhibited by a CTGF antisense oligodeoxynucleotide. Overexpression of CTGF by pcDNA3.1(+)/CTGF increased [(3)H]proline incorporation in cultured AFs. The results demonstrated that, in cultured AFs, Ang II increased CTGF production via AT(1)-R, which could be mediators of collagen synthesis by Ang II. This finding suggests that CTGF might be a novel target for antifibrotic therapy in vascular diseases.

Circulating endothelial progenitor cells, C-reactive protein and severity of coronary stenosis in Chinese patients with coronary artery disease.

We sought to investigate whether numbers and activity of circulating endothelial progenitor cells (EPCs) correlate with severity of coronary stenosis as well as cardiovascular risk factors in patients with stable coronary artery disease (CAD). Number of circulating EPCs was analyzed in 104 consecutive patients with proven or clinically suspected CAD. Adhesive and migratory activity was also determined. The number of EPCs was lower in patients with a single diseased coronary artery (Group II, n=35, p<0.05 vs. Group I) or multiple diseased arteries (Group III, n=25, p<0.01 vs. Group I, p<0.05 vs. Group II) compared to those with normal coronary arteries (Group I, n=44). The number of EPCs was also related with angiographic Gensini score (r=-0.355, p=0.006). In addition, concentrations of C-reactive protein (CRP) were elevated in patients with CAD, and positively correlated with Gensini score (r=0.476, p=0.001). As for the risk factors, the number of EPCs was also inversely correlated with age (p=0.001), high sensitivity-CRP (p=0.012), hypertension (p=0.042) and family history of CAD (p=0.043). Most importantly, the migratory capacity of EPCs was compromised in patients with CAD, and inversely correlated with the angiographic Gensini score (r=-0.315, p=0.021). EPCs isolated from patients with CAD also showed an impaired adhesive activity (p<0.05). In conclusion, in patients with stable CAD, reduction in the number and impairment in the function of circulating EPCs were correlated with the severity of coronary stenosis. CRP may play an important role in reducing the number of EPCs and accelerating atherosclerosis. Given the important role of EPCs in neovascularization of ischemic tissue, a decrease in the number and activity of EPCs may contribute to the impaired vascularization in patients with CAD.

Therapeutic angiogenesis by intramuscular injection of fibrin particles into ischaemic hindlimbs.

1. Fibrin gel has been used as a carrier of angiogenic molecules to promote neovascularization in animal models of limb ischaemia. However, little is known about the effects of fibrin itself under such pathological conditions. Accordingly, the present study tested the efficacy of fibrin in a rabbit model of acute hindlimb ischaemia. 2. Unilateral ischaemia was induced by resection of the left femoral artery. Seven days after surgery, fibrin particles (FP), which were free of fibrinogen, thrombin and vascular endothelial growth factor, were injected directly into the ischaemic thigh muscles. Twenty-four rabbits were divided into four groups, namely a control group receiving phosphate-buffered saline and three FP-treated groups receiving 5, 10 or 20 mg FP. 3. Collateral vessel development and limb perfusion were assessed by angiography, measuring the calf blood pressure ratio (BPR), thermographic scanning and the histological determination of capillary density. 4. At day 35 post-surgery, the treatment with 5 mg FP produced an augmentation of collateral vessel development (P < 0.01), increased numbers of capillaries (P < 0.05) and improved perfusion manifested by a higher blood flow (P < 0.01) and calf BPR (P < 0.05) compared with controls. Treatment with 10 and 20 mg FP had similar effects to those observed with 5 mg FP. 5. The present study reveals that FP promotes angiogenesis in a rabbit model of hindlimb ischaemia, thus providing a feasible approach to therapeutic angiogenesis in ischaemic diseases.

Therapeutic neovascularization by autologous transplantation with expanded endothelial progenitor cells from peripheral blood into ischemic hind limbs.

AIM: To investigate the hypothesis that transplantation with expanded autologous endothelial progenitor cells (EPC) could enhance neovascularization. METHODS: Peripheral blood mononuclear cells (PB-MNC) isolated from New Zealand White rabbits were cultured in vitro. At d 7, the adherent cells were collected for autologous transplantation. Rabbits with severe unilateral hind limb ischemia were randomly assigned to receive phosphate-buffered saline or expanded EPC in phosphate-buffered saline, administered by intramuscular injection in 6 sites of the ischemic thigh at postoperative d 7. Neovascularization was monitored by using the calf blood pressure ratio to indicate tissue perfusion, digital subtraction angiography to identify collateral vessel development and histological analysis of capillary density in the ischemic limb at d 35 after surgery. RESULTS: Autologous EPC transplantation produced significant amelioration in ischemic hind limbs, as indicated by a greater calf blood pressure ratio (0.52+/-0.04 vs 0.42+/-0.05, P<0.01), angiographic score (1.44+/-0.06 vs 0.98+/-0.08, P<0.01) and capillary density in muscle (195.2+/-5.4/mm2 vs 169.4+/-6.4/mm2, P<0.05), than controls. CONCLUSION: Transplantation of autologous expanded EPC can promote neovascularization in ischemic hindlimbs.

Differentiation of endothelial progenitor cells from human umbilical cord blood CD 34+ cells in vitro.

AIM: To study the time course of the expression of stem cell marker and endothelial cell markers on human cord blood CD34+ cells during in vitro differentiation process of endothelial progenitor cells (EPC). METHODS: CD34+ cells were selected and enriched from human cord blood by magnetically activated cell sorting (MACS), and cultured in dishes coated with or without fibronectin (Fn). Endothelial cells were identified by staining the cells with anti Flk-1 and vWF antibodies. The percentage of AC133+ cells in adherent CD34+ cell population was analyzed by fluorescence-activated cell sorting (FACS). RESULTS: The expression of Flk-1 and vWF on adherent CD34+ cells increased during the culture time, with 27.0 % positive for Flk-1 and negative for vWF at d 3, and 100 % positive for both Flk-1 and vWF at d 7. When cells were cultured in Fn-treated dishes, the percentages of Flk-1 and vWF positive cells increased to 34 % and 47 %, respectively at d 3, and 100 % at d 7. In contrast, the percentages of AC133+ cells among the adherent cell population decreased rapidly, and similar changes occurred in cells cultured in the presence of Fn. CONCLUSION: The gradual appearance of endothelial cell markers and the disappearance of stem cell marker characterized the in vitro differentiation of endothelial progenitor cells. Fibronectin accelerated the differentiation process of EPC.