Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2)

Tissue integration between a tissue-engineered corneal equivalent and the host eye is of critical importance in ensuring long-term implant success. A novel dendrimer cross-linked collagen scaffold has previously shown good corneal epithelial cell compatibility in vitro particularly when the highly functional dendrimer cross-linkers were functionalized to introduce additional biological groups. Herein we investigated heparinization of these materials and their potential to facilitate the delivery of basic fibroblast growth factor (FGF-2) in an active form, ultimately for use as a corneal tissue scaffold. Collagen gels cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry, and varying amounts of polypropyleneimine octaamine generation 2 (G2) dendimer and heparin were synthesized. Swelling studies and differential scanning calorimetry characterization indicated higher gel stability with the introduction of dendrimer cross-linking, which was not compromised by heparin integration. Dendrimer cross-linked gels with or without heparin gave multiple denaturation peaks, as did the heparinized EDC gels. This is thought to be the result of the heterogeneous cross-linking possible between collagen, the dendrimer and heparin. Release of FGF-2 from collagen gels showed typical first-order kinetics, with an initial burst followed by a prolonged gradual release. Heparinized dendrimer cross-linked gels released approx. 40% of the growth factor over a 2-week period, with significance maintenance of growth factor activity. Incorporation of heparin resulted in somewhat prolonged release from these systems.     

Mutations in SERPINF1 cause osteogenesis imperfecta type VI

Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the posttranslational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next‐generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium–derived factor. Hence, loss of pigment epithelium–derived factor function constitutes a novel mechanism for OI and shows its involvement in bone mineralization. © 2011 American Society for Bone and Mineral Research     

Protein A-immobilized microporous polyhydroxyethylmethacrylate affinity membranes for selective sorption of human-immunoglobulin-G from human plasma

Microporous membranes made of poly(2-hydroxyethylmethacrylate) [poly(HEMA)] carrying protein A were used for selective sorption of human-IgG from human plasma. Poly(HEMA) membranes were prepared by a photo-polymerization technique, and activated by cyanogen bromide (CNBr) in an alkaline medium (pH 11.5). Bioligand protein A was then immobilized by covalent binding onto these CNBr-activated membranes. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The non-specific adsorption of protein A on the plain poly(HEMA) membranes was 2.9 μg cm^-2. Maximum protein A immobilization was observed at pH 9.5. Up to 186 μg cm^-2 was immobilized on the CNBr-activated poly(HEMA) membranes. The maximum adsorption of human-IgG on the protein A-immobilized poly(HEMA) membranes was observed at pH 8.0. The non-specific adsorption of human-IgG onto the plain poly(HEMA) membranes was low (about 4.4 μg cm^-2). Higher human-IgG adsorption values (up to 394 μg cm^-2) were obtained in which the protein A-immobilized poly(HEMA) membranes were used. Much higher amounts of human-IgG (up to 489 μg cm^-2) were adsorbed from human plasma. Up to 91 % of the adsorbed human-IgG was desorbed by using 0.1 M aminoacetic acid as elution agent. The adsorption-desorption cycle was repeated ten times using the same polymeric membranes. There was no remarkable reduction in the adsorption capacity of the protein A-immobilized poly(HEMA) membranes.     

胆管癌微血管计数和VEGF及MMP2的表达及其意义

目的：探讨胆管癌组织中微血管密度（MVD）、血管内皮生长因子（VEGF）、基质金属蛋白酶2（MMP2）与胆管癌转移、预后的关系。方法：应用免疫组化的方法检测45例胆管癌及8例正常肝外胆管组织的MVD，VEGF和MMP2表达。结果：（1）胆管癌组织MVD值，VEGF，MMP2阳性表达率显著高于正常胆管组织（P＜0．05）；（2）胆管癌组织MVD，VEGF，MMP2的表达强度与胆管癌的病理分期、淋巴结转移有显著相关性（P＜0．05）；结论：MVD增高提示胆管癌转移侵袭潜能增加，VEGF，MMP2可能作为预测胆管癌转移的指标。     

青藤碱抗炎抗风湿作用机理研究

【目的】研究青藤碱抗炎、抗风湿作用机理。【方法】采用小鼠巨噬细胞株J774体外培养,观察药物对该细胞株增殖、一氧化氮(NO)合成以及肿瘤坏死因子-α信使核糖核酸(TNF-α mRNA)表达及生物合成的影响。【结果】青藤碱能够抑制该细胞株增殖,抑制脂多糖(LPS)刺激状态下的NO合成;对TNF-α蛋白合成具有抑制作用,但仅在高浓度时才对TNF-α mRNA表达具有轻微的抑制作用。【结论】青藤碱可能通过下调单核／巨噬细胞系统炎症介质和细胞因子合成发挥抗炎、抗风湿作用。     

The neurovascular unit and its growth factors: coordinated response in the vascular and nervous systems

Abstract

The nervous and vascular systems contain many common organizational features and develop similarly in terms of anatomical patterning. During embryogenesis and in regions of the brain undergoing postnatal neurogenesis, neural stem cells and endothelial cells are found in close proximity, or within a so-called vascular niche. The similarities in patterning and proximity may reflect coordinated development based on responsiveness to similar growth factors such as vascular endothelial growth factor, semaphorin, and ephrins/Ephs: molecules involved in the development and maintenance of both the nervous and vascular systems. Despite the blatant similarities between the vascular and nervous systems, little is still known about the co-dependence and/or interactions between the two systems during development and following alterations in metabolic demand as seen during aging, exercise, and disease processes. The interactions between the two systems involving common growth factors suggest these two systems have evolved in an interconnected way.     

Role of basic fibroblast growth factor in the course of cerebral vasospasm in an experimental model of subarachnoid hemorrhage

Abstract

The goal of this study was to investigate the relationship between basic fibroblast growth factor (bFGF) and the course of cerebral vasospasm after subarachnoid hemorrhage (SAH), using an immunohistochemical method. Female Sprague-Dawley rats were sacrificed by perfusion fixation 10 min, 6 h, 1, 2, 3, 4, 7 or 14 days after a single intracisternal injection of fresh autologous arterial blood. Morphometric analysis of lumen cross-sectional areas of blood vessels were determined by computerized image analysis. Results were expressed as percent lumen patency, defined as the ratio of the area of vessel patency in SAH rats to the area of patency in control rats. An immunohistochemical analysis against bFGF was performed using the avidin-biotin-peroxidase technique. The immuno-reactivity of bFGF was observed with the aid of a light microscope and semiquantitatively graded. Basilar arterial spasm was greatest 10 min after SAH (mean decrease: 67.1% of the control values; p < 0.001). Subsequently, there was a significant degree of spasm of the artery for three days after SAH, followed by full recovery at day 4. A slight increase in immunoreactivity was observed in the intima only at 10 min and one day after SAH. In the media, immunoreactivity showed a biphasic pattern; a significant increase in immunoreactivity was observed at 10 min that persisted for two days after SAH. At three days after SAH, immunoreactivity in the media returned to the control level, but then gradually increased significantly to reach a maximum at 14 days after SAH while the vascular dimensions were normal. Immunohistochemical analysis failed to show a direct relationship between bFGF and the course of cerebral vasospasm in this rat single-hemorrhage model. However, the late phase upregulation of bFGF might lead to the vascular angiopathy, fibrosis or hyperplasia during the chronic stage of SAH. [Neurol Res 2002; 24: 365-372]     

Vasoactive intestinal peptide influences neurite outgrowth in cultured rat spinal cord neurons

Abstract

Vasoactive intestinal peptide (VIP) is a neuropeptide which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro . For the purpose of clarifying the effect of VIP on spinal cord neurons, we studied the effect of VIP on neurite outgrowth of fetal rat ventral and dorsal portions of spinal cord in cultures. VIP-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had a significant neurite outgrowth at 10^-8, 10^-6, and 10^-4 M. The effect was considered to be concentration dependent. Morphological changes of the dorsal spinal cord cultures (DSCC) remained unchanged by VIP treatment. Because of their close sequence homology with VIP, PHI-27 (peptide, histidylisoleucine amide) and secretin were also examined with the same experimental conditions as was VIP. Both PHI-27 and secretin had neurite promoting effects in VSCC at 10^-8 and 10^-6 M, respectively. However, there were no neurite promoting effects in DSCC in both of them at any concentrations. VIP had the most potent effect on neurite outgrowth in VSCC, followed by PHI-27, and secretin in their effectiveness concentrations. Our data showing VIP, PHI-27 and secretin have neurotrophic action on VSCC and suggest that a potential therapeutic use of VIP and its related peptides in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis. [Neurol Res 2001; 23: 851-854]     

Angiopoietin 1 counteracts vascular endothelial growth factor-induced blood–brain barrier permeability and alleviates ischemic injury in the early stages of transient focal cerebral ischemia in rats

Abstract

Objective: To examine the effects of intraventricular pre-treatment with a combination of recombinant adeno-associated virus vectors encoding VEGF (rAAV-VEGF) and Ang1 (rAAV-Ang1) on early stroke in a rat model of transient middle cerebral artery occlusion (tMCAO).

Methods: rAAV-VEGF/rAAV-Ang1 or rAAV-VEGF/rAAV-null vector was delivered into the lateral ventricles of 48 rats. Eight weeks later, the rats were subjected to tMCAO for 2 hours. During the early stages of ischemic reperfusion, VEGF and Ang1 expression levels, blood–brain barrier (BBB) permeability and cerebral microvessel density were determined and compared statistically between groups. Cerebral infarct volume and modified neurological severity scores (NSS) were also determined to evaluate the therapeutic efficacy of rAAV-VEGF/rAAV-Ang1.

Results: Intraventricular application of rAAV-VEGF/rAAV-Ang1, 8 weeks before tMCAO, resulted in VEGF and Ang1 overexpression, and significantly decreased Evans blue permeability following ischemia (p<0.05). The microvessel density in the peri-infarct zone was significantly increased in the rAAV-VEGF/rAAV-Ang1 group compared with the rAAV-VEGF/rAAV-null group (p<0.05). Cerebral infarct volume and NSS in the rAAV-VEGF/rAAV-Ang1 group were significantly decreased compared with the rAAV-VEGF/rAAV-null group (p<0.05).

Conclusion: In cerebral ischemia, the combination of Ang1 and VEGF could be used early to promote the formation of mature neovessels and protect the injured cells, without inducing the side effects on BBB permeability. Early intraventricular injection of mixed rAAV-VEGF and rAAV-Ang1 may be a favorable therapeutic strategy in gene therapy for experimental stroke.     

Low bone mass and high material bone density in two patients with Loeys-Dietz syndrome caused by transforming growth factor beta receptor 2 mutations

Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by heterozygous mutations in the genes encoding transforming growth factor beta receptor 1 or 2 (TGFBR1 or TGFBR2). Although an association between LDS and osteoporosis has been reported, the skeletal phenotype regarding bone mass is not well characterized. Here, we report on two LDS patients with mutations in TGFBR2. Patient 1 was a 24-year-old man who had a total of three fractures involving the left radius, the left metacarpal, and the right femur. At the age of 14 years, lumbar spine areal bone mineral density Z-score was -4.0 and iliac bone histomorphometry showed elevated bone turnover (bone formation rate per bone surface: 91 μm3/μm2/year; age-matched control values 37 [10], mean [SD]) and mildly low trabecular bone volume per tissue volume (17.2%; age-matched control values 25.7 [5.3]). Bone mineralization density distribution (BMDD) in trabecular bone was increased (Ca(Peak) 22.70 wt% Ca; age-matched control values 21.66 [0.52]). Patient 2, a 17-year-old girl, suffered from diffuse bone pain but had not sustained fractures. At 14 years of age, her lumbar spine areal bone mineral density Z-score was -3.4. Iliac bone histomorphometry at that age confirmed low bone mass (bone volume to tissue volume 10.1%, same control values as above) and high bone turnover (bone formation rate per bone surface 70 μm3/μm2/year). BMDD in trabecular bone was significantly shifted toward increased mineralization (Ca(Peak) 22.36 wt% Ca). Thus, it appears that LDS can be associated with low bone mass and high bone turnover but increased matrix mineralization of trabecular bone.