Engineering angiogenesis following spinal cord injury: a coculture of neural progenitor and endothelial cells in a degradable polymer implant leads to an increase in vessel density and formation of the blood–spinal cord barrier

Angiogenesis precedes recovery following spinal cord injury and its extent correlates with neural regeneration, suggesting that angiogenesis may play a role in repair. An important precondition for studying the role of angiogenesis is the ability to induce it in a controlled manner. Previously, we showed that a coculture of endothelial cells (ECs) and neural progenitor cells (NPCs) promoted the formation of stable tubes in vitro and stable, functional vascular networks in vivo in a subcutaneous model. We sought to test whether a similar coculture would lead to the formation of stable functional vessels in the spinal cord following injury. We created microvascular networks in a biodegradable two-component implant system and tested the ability of the coculture or controls (lesion control, implant alone, implant + ECs or implant + NPCs) to promote angiogenesis in a rat hemisection model of spinal cord injury. The coculture implant led to a fourfold increase in functional vessels compared with the lesion control, implant alone or implant + NPCs groups and a twofold increase in functional vessels over the implant + ECs group. Furthermore, half of the vessels in the coculture implant exhibited positive staining for the endothelial barrier antigen, a marker for the formation of the blood–spinal cord barrier. No other groups have shown positive staining for the blood–spinal cord barrier in the injury epicenter. This work provides a novel method to induce angiogenesis following spinal cord injury and a foundation for studying its role in repair.     

改良单宁酸-氯化铁法媒染子宫血管的实验研究

目的：光镜下观察大鼠子宫各部位血管构筑。方法：应用单宁酸一氯化铁法(TA—Fe法)灌流固定大鼠，取子宫不同位置做冰冻切片，氯化铁显色，常规脱水、透明、封片，显微摄影。结果：子宫壁出现类似电镜负染色效果，组织结构灰黑色，血管双线条状，分支明显，过管壁切面可见内皮细胞或平滑肌；子宫动脉进入肌层分支并形成一、二级血管网和多支环状动脉，环行动脉或二级血管网发出的微动脉和毛细血管进入粘膜，形成密集的三级毛细血管网。结论：经灌流的子宫标本较长时间保存在媒染固定液内，导致血管外结构也被媒染；子宫壁有三级血管网；每个子宫有多支环状动脉；各段子宫血管构筑不尽相同。     

Expression of the endothelial markers PECAM-1, vWf,and CD34 in vivo and in vitro

EC culture models are essential to study pathological alterations of endothelial cells (ECs) in pulmonary vascular diseases under standardized conditions. Nevertheless, little is known about the spectrum of alterations of vessel-specific endothelial phenotypes in monolayer cultures. For the comparative study of endothelial markers in vivo and in vitro we investigated immunohistochemically the expression of PECAM-1, vWf, and CD34 by pulmonary ECs in vivo and in stimulated/unstimulated human umbilical vein endothelial cells (HU-VEC) and human pulmonary microvascular endothelial cells (HPMEC). In vivo, vessel type-specific expression patterns were found for vWf and CD34, while PECAM-1 was homogeneously and strongly expressed. While all HUVEC showed a marked vWf staining, about two-thirds of HPMEC exhibited a strong and the rest a moderate vWf staining. In both in vitro models all ECs were clearly PECAM-1-positive. However, only about 20% of the HUVEC and HPMEC were CD34-positive. Our results demonstrate the reduced expression of vessel type-specific endothelial phenotypes by endothelial monolayer cultures, stressing the need to improve culture conditions as well as develop cocultures and three-dimensional culture models. Moreover, the need for endothelial markers specific for single microvascular type ECs becomes obvious in order to establish cultures consisting of only one microvascular ECs subpopulation.     

Microvasculature of the buffalo epididymis

The microvasculature of the water buffalo (Bubalus bubalis) epididymis was investigated using light (LM), scanning electron (SEM), and transmission electron (TEM) microscopy techniques. SEM analysis of the buffalo epididymis showed fenestrations that occupied ovoid inside the endothelium of the postcapillary venules located in the caput, corpus, and cauda. They varied in shape and dimension, but more importantly, they connected the venules of the blood vascular system to the capillaries of the peripheral lymphatic vascular system. Morphofunctional analysis of these connections suggests that the microvasculature of the buffalo epididymis plays a role in facilitating the circulation of biologically active substances, and the absorption and secretion processes necessary for the survival and maturation of spermatozoa. The lymphatic capillaries at the connection points formed a network of variously sized polygonal links. These capillaries then converged to form the precollector lymphatic vessels, which in turn converged with the larger vessels originating from the testis. It was further noted that in the capillary endothelium there were no fenestrations, and in the large veins there were many diverticula. These diverticula appear to play a role in the regulation of the seasonal variations of the blood reflux. In general, the microvascular architecture of the buffalo epididymis, particularly its connection to the lymphatic vascular system, appears to play an important role in the absorption and secretion processes of the epididymal epithelium. Anat Rec 266:58–68, 2002. © 2002 Wiley‐Liss, Inc.     

Endothelial-Derived miR-17∼92 Promotes Angiogenesis to Protect against Renal Ischemia-Reperfusion Injury

BackgroundDamage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)–mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established.MethodsAntibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92^endo−/−) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated.ResultsmiR-17, -18a, -20a, -19b, and pri–miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92^endo−/− exacerbates renal IRI in male and female mice. Specifically, miR-17∼92^endo−/− promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92^endo−/− after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate.ConclusionsThese data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.     

胎儿房室结微血管构筑的扫描电镜观察

用血管铸型结合扫描电镜方法观察了7例胎儿房室结微血管构筑.微动脉反复分支呈树状,行走方式呈波浪形或螺旋形.毛细血管网可分为内外两层:外层纵行为主,以"H"或"Y"形吻合;内层弯曲行走为主,并互相交错吻合.以房室结动脉主干为界,其下方毛细血管密度较大,其上方及前方较小.房室结的上方、前下方和后下方均有微动脉连接毛细血管网.整个结的毛细血管大致可分为结后部上方、下方及前部三个区域.靠毛细血管网外层处可见管径较粗、呈袋状的微静脉.     

幼儿外侧膝状体微血管的形态学分析

目的分析幼儿外侧膝状体微血管的形态结构。方法采用8例死亡24 h内的幼儿外侧膝状体,经碱性磷酸酶染色、光镜观察及体视学处理,对幼儿外侧膝状体微血管密度进行分析。结果幼儿外侧膝状体后2/3部微血管密度高于前1/3部的微血管密度,经t检验,P<0.01。结论①幼儿阶段外侧膝状体与其相对应的视皮质各区微血管密度的大小相一致。②按照功能与结构相一致的原则,本课题研究结果支持“直接兴奋性输入会聚学说”。     

Assessment of Angiogenesis and Cell Survivability of an Inkjet Bioprinted Biological Implant in an Animal Model

The rapidly growing field of tissue engineering hopes to soon address the shortage of transplantable tissues, allowing for precise control and fabrication that could be made for each specific patient. The protocols currently in place to print large-scale tissues have yet to address the main challenge of nutritional deficiencies in the central areas of the engineered tissue, causing necrosis deep within and rendering it ineffective. Bioprinted microvasculature has been proposed to encourage angiogenesis and facilitate the mobility of oxygen and nutrients throughout the engineered tissue. An implant made via an inkjet printing process containing human microvascular endothelial cells was placed in both B17-SCID and NSG-SGM3 animal models to determine the rate of angiogenesis and degree of cell survival. The implantable tissues were made using a combination of alginate and gelatin type B; all implants were printed via previously published procedures using a modified HP inkjet printer. Histopathological results show a dramatic increase in the average microvasculature formation for mice that received the printed constructs within the implant area when compared to the manual and control implants, indicating inkjet bioprinting technology can be effectively used for vascularization of engineered tissues.     

Red grape (Vitis vinifera L.) flavonoids down‐regulate collagen type III expression after UV‐A in primary human dermal blood endothelial cells

Red grape (Vitis vinifera L.) flavonoids including flavan‐3‐ols (eg, catechin and epicatechin), flavonols (eg, quercetin) and anthocyanins (eg, malvidin) exert anti‐inflammatory and antioxidant activities. In the skin they also have a photoprotective action, and their effects have been extensively investigated in keratinocytes, melanocytes and fibroblasts. Despite their known effects also on blood vasculature, little is known on their activities on human dermal blood endothelial cells (HDBECs), which are critically involved in skin homeostasis as well as in the pathogenesis of neoplastic and inflammatory skin diseases. We sought to study the biological effects of selected red grape flavonoids in preventing the consequences of ultraviolet (UV)‐A irradiation in vitro. Our results show that red grape flavonoids prevent UV‐A‐induced sICAM‐1 release in HDBECs, suggesting that this cell type could represent an additional target of the anti‐inflammatory activity of flavonoids. In addition, flavonoids effectively inhibited UV‐A‐induced synthesis of collagen type III at both RNA and protein level, indicating that dermal blood microvasculature could be actively involved in ECM remodelling as a consequence of skin photo‐ageing, and that this can be prevented by red grape flavonoids.