Roberto F. Nicosia

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel, collagen, fibrin, and plasma clot

SummaryRings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of3H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis.

Delayed Graft Function and Cast Nephropathy Associated with Tacrolimus Plus Rapamycin Use

Delayed graft function (DGF) occurs in 15 to 25% (range, 10 to 62%) of cadaveric kidney transplant recipients and up to 9% of living donor recipients. In addition to donor, recipient, and procedural factors, the choice of immunosuppression may influence the development of DGF. The impact of immunosuppression on DGF was studied. The frequency of DGF was evaluated in first cadaveric or living donor kidney allograft recipients (n = 144) transplanted at the University of Washington from November 1999 through September 1, 2001. Donor, recipient, and procedural factors, as well as biopsy results, were compared between patients who developed DGF and those who did not. DGF was more common in patients treated with rapamycin than without (25% versus 8.9%, P = 0.02) and positively correlated with rapamycin dose (P = 0.008). In those developing DGF, the duration of posttransplant dialysis increased with donor age (P = 0.003) but decreased with mycophenolate mofetil use (P = 0.01). All biopsies during episodes of DGF demonstrated changes of acute tubular injury. Of the patients with tubular injury, 12 treated with rapamycin and tacrolimus developed intratubular cast formation indistinguishable from myeloma cast nephropathy. Histologic, immunohistochemical, and ultrastructural studies indicated that these casts were composed at least in part of degenerating renal tubular epithelial cells. These findings suggest that rapamycin therapy exerts increased toxicity on tubular epithelial cells and/or retards healing, leading to an increased incidence of DGF. Additionally, rapamycin treatment combined with a calcineurin inhibitor may lead to extensive tubular cell injury and death and a unique form of cast nephropathy.

Histotypic angiogenesis in vitro: Light microscopic, ultrastructural, and radioautographic studies

SummaryA model for the study of angiogenesis in vitro is described. Rat aortas, cultured in a tridimensional matrix of clotted chick plasma, gave rise to luxuriant outgrowth of vascular channels. We studied this process with light microscopic, radioautographic, and ultrastructural techniques. On the 2nd d of culture, endothelial cells sprouted from the intima of the aorta and its collateral branches into the surrounding clot, forming solid cellular cords. A complex vascular network was established within the 1st wk by spindly, poorly differentiated endothelial cells. At this stage cells were migrating, branching, and proliferating in a longitudinal fashion (labeling index: 67.4% ± 7.7). Lumens, when present, appeared as slitlike spaces enclosed with junctional complexes. By the end of the 2nd wk the migratory activity decreased and proliferation occurred mostly in a cross-sectional plane, with formation of large patent lumens (labeling index: 48% ± 3.1). Vascular channels were lined by prominent endothelial cells rich in rough endoplasmic reticulum, polysomes, mitochondria, Golgi apparatuses, and coated vesicles. Cells were enveloped with a ruthenium red positive layer, particularly abundant on the luminal surface and in the interendothelial space. A discontinuous basal lamina was present along the abluminal side. At 28 d the labeling index was reduced to 2.25% ± 0.9. The still viable endothelium exhibited numerous microfilaments and microtubules, decreased cytoplasmic organelles, and increased pinocytotic activity. This experimental model, histophysiologic gradient culture, provides us with a new tool for the study of vascular morphogenesis, angiogenesis dependent growth of tumors, and neoplastic intravasation.

Regulation of Vascular Growth and Regression by Matrix Metalloproteinases in the Rat Aorta Model of Angiogenesis

Matrix metalloproteinases (MMPs) have been implicated in the formation of microvessels during angiogenesis, but their role in vascular regression is poorly understood. The rat aorta model of angiogenesis was used to study the function of MMPs at different stages of the angiogenic process. Gelatin zymography and Western analysis demonstrated production of MMP-2 and MMP-9 by aortic outgrowths in serum-free collagen gel culture. MMP-2 was found in both culture medium and collagen gel, whereas MMP-9 was predominantly associated with the gel. MMP expression increased gradually during the angiogenic growth phase and stayed high when vessels regressed and collagen lysed around the aortic rings. The MMP inhibitors, batimastat and marimastat, blocked formation of microvessels when added to the culture medium at the beginning of the experiment. They, however, stabilized the microvessels and prevented vascular regression after the angiogenic growth phase. This effect was observed also under conditions of angiogenic stimulation by basic fibroblast growth factor. MMP inhibitor-mediated stabilization of microvessels was associated with inhibition of collagen lysis and accumulation of collagen fibrils in the subendothelial space. This study demonstrates that MMPs promote microvessel formation during the early stages of angiogenesis, but also contribute to the reabsorption of the neovasculature in the later stages of this process. The time-dependent divergent effects of MMPs on microvessel growth and survival may influence the in vivo activity of MMP inhibitors used to treat angiogenesis-dependent disorders.

Chapter 7 The Aortic Ring Model of Angiogenesis

Angiogenesis is regulated by a complex cascade of cellular and molecular events. The entire process can be reproduced in vitro by culturing rat or mouse aortic explants in three-dimensional biomatrices under chemically defined conditions. Angiogenesis in this system is driven by endogenous growth factors released by the aorta and its outgrowth. Sprouting endothelial cells closely interact with pericytes, macrophages, and fibroblasts in an orderly sequence of morphogenetic events that recapitulates all stages of angiogenesis. This model can be used to study the basic mechanisms of the angiogenic process and to test the efficacy of proangiogenic or antiangiogenic compounds. Aortic cultures can be evaluated with a range of morphologic and molecular techniques for the study of gene expression. In this chapter we describe basic protocols currently used in our laboratory to prepare, quantify, and analyze this assay.

Regulation of angiogenesis by vascular endothelial growth factor and angiopoietin-1 in the rat aorta model: distinct temporal patterns of intracellular signaling correlate with induction of angiogenic sprouting.

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) promote the spontaneous angiogenic response of freshly cut rat aortic rings. When VEGF and Ang-1 were tested in cultures of 14-day-old rings, which are quiescent and unable to spontaneously produce neovessels, only VEGF was capable of inducing an angiogenic response. Ang-1 failed to initiate angiogenesis in this system, but significantly potentiated VEGF-induced neovessel sprouting. Potential differences in cell signaling triggered by VEGF and Ang-1 were evaluated in cultures of quiescent rings. VEGF induced biphasic and prolonged (15 minutes and 4 to 24 hours) phosphorylation of p44/42 MAPK and Akt, while the effect of Ang-1 was transient and monophasic (15 minutes). Both VEGF and Ang-1 induced rapid, monophasic (15 minutes) phosphorylation of p38 MAPK. When VEGF and Ang-1 were administered together, the second peak of VEGF-induced p44/42 MAPK phosphorylation was markedly reduced. The effect of the VEGF/Ang-1 combination on AKT phosphorylation was, instead, additive over time, and sustained over a 24-hour period. The VEGF/Ang-1 combination caused an additive effect also on p38 MAPK phosphorylation at 1 hour. Confocal microscopy of VEGF-, Ang-1, or VEGF/Ang-1-stimulated aortic rings double stained at time points of maximal phosphorylation for cell markers and signal transduction proteins demonstrated phosphorylated p44/42 MAPK, p38 MAPK, and Akt predominantly in endothelial cells. Experiments with specific inhibitors demonstrated that p44/42 MAPK and Akt, but not p38 MAPK, are necessary for neovessel sprouting. These results identify p44/42 MAPK and Akt as critical intracellular mediators of angiogenesis, whose transient phosphorylation is, however, not sufficient for the initiation of this process. The observation that sustained phosphorylation of these signaling pathways, particularly of Akt, correlates with induction of angiogenesis suggests that the duration of phosphorylation signals influences critical cellular events required for the induction of angiogenic sprouting.

Human vasculogenesis ex vivo: embryonal aorta as a tool for isolation of endothelial cell progenitors.

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11- to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1;[sol;vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD31−), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34+/CD31+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34+/CD31− endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.

The thin prep rat aortic ring assay: A modified method for the characterization of angiogenesis in whole mounts

The rat aortic ring model has gained broad acceptance as an angiogenic assay. This system can be used to study the activity of angiogenic and anti-angiogenic factors, and investigate the molecular mechanisms of the angiogenic process. We describe here a thin prep modification of the aortic ring model, which significantly simplifies the procedure and allows staining of aortic outgrowths as whole mounts. Using this procedure, intact preparations of angiogenic outgrowths are successfully and reproducibly stained with endothelial cell (anti-CD-31 and -Tie2 antibodies, Griffonia Simplicifolia isolectin-B4) and smooth muscle cell (anti-α-smooth muscle actin antibody) markers. Combined use of double immunostaining and confocal microscopy allows concurrent visualization of endothelial and mural cells in the same cultures. Whole mount immunostains of rat aorta cultures are an effective way to rapidly characterize the cellular composition of the angiogenic outgrowths, and localize proteins implicated in the regulation of angiogenesis. This method should facilitate the work of the many vascular biologists that have adopted the rat aorta model as a tool to study angiogenesis and its mechanisms.

The cytoskeletal framework of sea urchin eggs and embryos: Developmental changes in the association of messenger RNA☆

Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity.

SummaryRecent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epithelioid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.