Diabetes impairs arterio-venous specification in engineered vascular tissues in a perivascular cell recruitment-dependent manner.

Cell-based tissue engineering is a potential treatment alternative for organ replacement. However, the lack of a robust vasculature, especially in the context of diseases such as diabetes, is a major hindrance to its success. Despite extensive research on the effects of diabetes in angiogenic sprouting, its effects on vessel arterio-venous (AV) specification have not been addressed. Using an engineered tissue that yields functional vessels with characteristic AV identities, we demonstrate that type 1 diabetes negatively affects vessel AV specification and perivascular cell (PVC) coverage. Blockage of PVC recruitment in normoglycemia does not affect blood flow parameters, but recapitulates the vascular immaturity found in diabetes, suggesting a role for PVCs in AV specification. The downregulation of Jagged1 and Notch3, key modulators of endothelial-perivascular interaction, observed in diabetes support this assertion. Co-culture assays indicate that PVCs induce arterial identity specification by inducing EphrinB2 and downregulating EphB4. This is antagonized by high glucose or blockage of endothelial Jagged1. Engineered tissues composed of microvessels from diabetic mice display normal PVC coverage and Jagged1/Notch3 gene expression when implanted into non-diabetic hosts. These indicate a lack of legacy effect and support the use of a more aggressive treatment of diabetes in patients undergoing revascularization therapies.

An endogenously deposited fibrin scaffold determines construct size in the surgically created arteriovenous loop chamber model of tissue engineering.

BACKGROUND: An arteriovenous loop (AVL) enclosed in a polycarbonate chamber in vivo, produces a fibrin exudate which acts as a provisional matrix for the development of a tissue engineered microcirculatory network. OBJECTIVES: By administering enoxaparin sodium - an inhibitor of fibrin polymerization, the significance of fibrin scaffold formation on AVL construct size (including the AVL, fibrin scaffold, and new tissue growth into the fibrin), growth, and vascularization were assessed and compared to controls. METHODS: In Sprague Dawley rats, an AVL was created on femoral vessels and inserted into a polycarbonate chamber in the groin in 3 control groups (Series I) and 3 experimental groups (Series II). Two hours before surgery and 6 hours post-surgery, saline (Series I) or enoxaparin sodium (0.6 mg/kg, Series II) was administered intra-peritoneally. Thereafter, the rats were injected daily with saline (Series I) or enoxaparin sodium (1.5 mg/kg, Series II) until construct retrieval at 3, 10, or 21 days. The retrieved constructs underwent weight and volume measurements, and morphologic/morphometric analysis of new tissue components. RESULTS: Enoxaparin sodium treatment resulted in the development of smaller AVL constructs at 3, 10, and 21 days. Construct weight and volume were significantly reduced at 10 days (control weight 0.337 +/- 0.016 g [Mean +/- SEM] vs treated 0.228 +/- 0.048, [P < .001]: control volume 0.317 +/- 0.015 mL vs treated 0.184 +/- 0.039 mL [P < .01]) and 21 days (control weight 0.306 +/- 0.053 g vs treated 0.198 +/- 0.043 g [P < .01]: control volume 0.285 +/- 0.047 mL vs treated 0.148 +/- 0.041 mL, [P < .01]). Angiogenesis was delayed in the enoxaparin sodium-treated constructs with the absolute vascular volume significantly decreased at 10 days (control vascular volume 0.029 +/- 0.03 mL vs treated 0.012 +/- 0.002 mL [P < .05]). CONCLUSION: In this in vivo tissue engineering model, endogenous, extra-vascularly deposited fibrin volume determines construct size and vascular growth in the first 3 weeks and is, therefore, critical to full construct development.

Exercise-induced arteriovenous intrapulmonary shunting in dogs.

RATIONALE: We have previously shown, using contrast echocardiography, that intrapulmonary arteriovenous pathways are inducible in healthy humans during exercise; however, this technique does not allow for determination of arteriovenous vessel size or shunt magnitude. OBJECTIVES: The purpose of this study was to determine whether large-diameter (more than 25 microm) intrapulmonary arteriovenous pathways are present in the dog, and whether exercise recruits these conduits. METHODS: Through the right forelimb, 10.8 million 25-microm stable isotope-labeled microspheres (BioPAL, Inc., Worcester, MA) were injected either at rest (n = 8) or during high-intensity exercise (6- 8 mph, 10-15% grade, n = 6). Systemic arterial blood was continuously sampled during and for 3 minutes after injection. After euthanasia, tissue samples were obtained from the heart, liver, kidney, and skeletal muscle. In addition, 25- and 50-microm microspheres were infused into four isolated dog lungs that were ventilated and perfused at constant pressures similar to exercise. MEASUREMENTS AND MAIN RESULTS: Blood and tissue samples were commercially analyzed for the presence of microspheres. No microspheres were detected in the arterial blood or tissue samples from resting dogs. In contrast, five of six exercising dogs showed evidence of exercise-induced intrapulmonary arteriovenous shunting, as microspheres were detected in arterial blood and/or tissue. Furthermore, shunt magnitude was calculated to be 1.4 +/- 0.8% of cardiac output (n = 3). Evidence of intrapulmonary arteriovenous anastomoses was also found in three of four isolated lungs. CONCLUSIONS: Consistent with previous human findings, these data demonstrate that intrapulmonary arteriovenous pathways are functional in the dog and are recruited with exercise.

A new ex vivo model to study venous angiogenesis and arterio-venous anastomosis formation.

Explants of rat inferior vena cava embedded in collagen gel and cultured under serum-free conditions produced microvascular outgrowths composed of endothelial cells and pericytes. Exogenous vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently stimulated angiogenesis and induced the formation of complex networks of highly branched microvessels. VEGF and the VEGF/bFGF combination also promoted pericyte recruitment. Medium conditioned by untreated vena cava cultures contained endogenous VEGF, and a blocking antibody against VEGF significantly reduced the spontaneous angiogenic response of the explants. Vena cava explants exhibited a greater capacity to form neovessels than aortic rings when tested in parallel cultures from the same animal. When compared with aorta-derived microvessels, neovessels of vena cava origin were longer and had fewer pericytes. Vena cava-aorta cocultures produced extensive anastomosing networks of microvessels, which were primarily contributed by the venous explants. Because of its florid angiogenesis and exquisite sensitivity to angiogenic factor stimulation, the vena cava model may provide novel insights into the regulation of the angiogenic process, which typically initiates from the venous side of the vascular bed. Combined with the aortic ring model, this new assay may also enhance our understanding of the mechanisms of anastomosis formation between the arterial and the venous circulations.

Postnatal development of the vascular pattern in the rat telencephalic pia-arachnoid. A SEM study.

The postnatal changes in arrangement of the vascular system of the pia-arachnoid of rats are described based on scanning electron microscopy of microcorrosion casts and transmission electron microscopy. At birth, the distal arteries and veins are embedded in a dense plexiform network of immature capillaries. Arteries and veins are interconnected by many small capillary anastomoses. The trunks are located above the pial plexus. The underlying plexiform vessels provide the matrix for the formation of additional collateral and precortical segments during further development. During the first postnatal week, the distal pial arteries and veins become visible as separate channels and emerge from the subjacent capillary plexus. The pattern of anastomosing arterial rings is now clearly visible. The pial arterial tree can be subdivided into conductive, collateral, and precortical distributive segments, according to Jokelainen et al. (1982). Subsequently, passive expansion of the vascular system takes place during the period of rapid brain growth. In young adults the majority of the formerly closed arterial rings are interrupted, possibly by regression of single collateral arterial segments (Fig. 6). The dense venous capillary plexus of the pia is maintained during the first eight days in spite of marked brain growth. The process of reduction of this capillary plexus starts at the arterial side and proceeds from proximal to distal segments of the veins during the second and third week. The capillary segments, which provide anastomosis between arterial and venous vessels, disappear at the same time as the regression of the dense venous capillary network.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologic fluorescein angiographic, and light microscopic features of experimental choroidal neovascularization.

We induced choroidal neovascularization in the rhesus monkey by impoverishing the blood supply to the inner retina and producing defects in Bruch's membrane by photocoagulation. Fourteen of 46 eyes undergoing photocoagulation developed neovascular fronds which were identified and categorized by histopathologic examination and fluorescein angiography. All new vessels gained access to the retina through defects in Bruch's membrane at the site of photocoagulation marks. In eight eyes the new vessels remained localized to the immediate vicinity of photocoagulation marks. In four eyes neovascular fronds infiltrated the subretinal space for distances up to 6 disk diameters from the point of entry into the retina. In the two eyes choroidovitreal neovascular complexes developed but rapidly regressed shortly after gaining the vitreous cavity. Fluorescein angiography demonstrated that all neovascular fronds were grossly incompetent to dye but that formed feeding channels had some degree of integrity. Light microscopic studies showed the proliferating networks to be composed of capillaries with well-formed basement membranes and more mature vessels with the basic structure of choroidal arteries and veins.