Endothelial cell promotion of early liver and pancreas development.

Different steps of embryonic pancreas and liver development require inductive signals from endothelial cells. During liver development, interactions between newly specified hepatic endoderm cells and nascent endothelial cells are crucial for the endoderm's subsequent growth and morphogenesis into a liver bud. Reconstitution of endothelial cell stimulation of hepatic cell growth with embryonic tissue explants demonstrated that endothelial signalling occurs independent of the blood supply. During pancreas development, midgut endoderm interactions with aortic endothelial cells induce Ptf1a, a crucial pancreatic determinant. Endothelial cells also have a later effect on pancreas development, by promoting survival of the dorsal mesenchyme, which in turn produces factors supporting pancreatic endoderm. A major goal of our laboratory is to determine the endothelial-derived molecules involved in these inductive events. Our data show that cultured endothelial cells induce Ptf1a in dorsal endoderm explants lacking an endogenous vasculature. We are purifying endothelial cell line product(s) responsible for this effect. We are also identifying endothelial-responsive regulatory elements in genes such as Ptf1a by genetic mapping and chromatin-based assays. These latter approaches will allow us to track endothelial-responsive signal pathways from DNA targets within progenitor cells. The diversity of organogenic steps dependent upon endothelial cell signalling suggests that cross-regulation of tissue development with its vasculature is a general phenomenon.

Fetal tracheal reconstruction with cartilaginous grafts engineered from mesenchymal amniocytes.

BACKGROUND/PURPOSE: This study was aimed at determining whether cartilaginous grafts engineered from mesenchymal cells normally present in the amniotic fluid could be used in fetal tracheal repair. METHODS: Ovine mesenchymal amniocytes were expanded in culture, labeled with green fluorescent protein, and seeded onto biodegradable scaffold tubes maintained in chondrogenic medium. After chondrogenic differentiation of the constructs was confirmed, they were used to repair either partial or full circumferential tracheal defects in allogeneic fetal lambs (n = 7). Newborns were evaluated for signs of airway compromise. Implants were harvested over a 10-day period postnatally for multiple analyses. RESULTS: All 5 lambs that survived to term were able to breathe spontaneously at birth, 4 (80%) of them without stridor. However, variable degrees of stridor developed over time in all but one animal. Mild-to-moderate tracheal stenosis was present in all specimens. Histologically, grafts contained green fluorescent protein-positive cells, were lined with pseudostratified columnar epithelium, and remodeled into a predominantly fibrous cartilage pattern. Implants showed no significant changes in glycosaminoglycans, collagen, and elastin content at harvest. CONCLUSIONS: Engineered cartilaginous grafts derived from mesenchymal amniocytes may become a viable alternative for tracheal repair. The amniotic fluid can be a practical cell source for engineered tracheal reconstruction.

An injectable tissue-engineered embolus prevents luminal recanalization after vascular sclerotherapy.

BACKGROUND/PURPOSE: Sclerotherapy for vascular malformations is often limited by luminal recanalization. This study examined whether an injectable tissue-engineered construct could prevent this complication in a rabbit model of venous sclerotherapy. METHODS: Ethanol sclerotherapy of a temporarily occluded jugular vein segment was performed in 46 rabbits, which were then divided into 3 groups. Group I (n = 16) had no further manipulations. In groups II (n = 15) and III (n = 15), 0.5 mL collagen hydrogel was injected intraluminally, respectively, devoid of and seeded with autologous fibroblasts. At 1, 4, and 20 to 24 weeks postoperatively, vein segments were examined for patency and resected for histological evaluation. Statistical analysis was by Fisher's Exact test. RESULTS: All vein segments were occluded at 1 and 4 weeks in all groups, despite histological evidence of progressive endothelial ingrowth. However, at 20 to 24 weeks, angiography demonstrated restoration of vessel patency in groups I (3/6) and II (3/5), but not in group III (0/6; P = .043), in which histology confirmed an obliterated lumen for all vessels. CONCLUSION: An injectable, fibroblast-based, engineered construct prevents midterm to long-term recanalization in a leporine model of vascular sclerotherapy. This novel therapeutic approach may prevent recurrence of vascular malformations after sclerotherapy, thus reducing the need for repeated procedures and morbid operative resections.

CDK2 translational down-regulation during endothelial senescence.

Here we report for the first time that loss of CDK2 activity, by translational inhibition and through CDK2 inhibition by p21(Cip1/Waf1), may be responsible for endothelial senescence. We show that expression of dominant-negative p53 extends human umbilical vein endothelial cell (HUVEC) lifespan past senescence. HUVEC expressing telomerase can completely bypass senescence and become immortal (i-HUVEC). Surprisingly, early passage i-HUVEC, like senescent HUVEC, express high levels of the CDK inhibitors p16(INK4a) and p21(Cip1/Waf1). Expression of p16(INK4a) can persist for over 280 population doublings, while p21(Cip1/Waf1) expression was eventually lost in five of six i-HUVEC lines. Senescent HUVEC contain undetectable CDK2 activity, which results from a dramatic reduction of CDK2 protein levels and inhibition of remaining CDK2 by p21(Cip1/Waf1). The decreased CDK2 levels in senescent HUVEC are not due to decreased transcription or protein stability; rather, CDK2 translation declines during senescence. Bypass of endothelial senescence by telomerase entails the restoration of CDK2 translation and activity. These results suggest that p16(INK4a) does not play a role in endothelial senescence. Rather, CDK2 translational down-regulation may be a key regulatory event in replicative senescence of endothelial cells. Understanding the mechanisms regulating endothelial senescence will be critical in determining the role of endothelial senescence in tumor growth.

Enhancement of intravascular sclerotherapy by tissue engineering: short-term results.

BACKGROUND/PURPOSE: Treatment of vascular malformations with sclerotherapy is often complicated by reexpansion secondary to endothelial recanalization. This study examined the use of an autologous fibroblast construct to enhance intraluminal scar formation after sclerotherapy. METHODS: New Zealand rabbits (n = 15) underwent ethanol sclerotherapy of a segment of the facial vein. After intraluminal saline flush, animals were equally divided into 3 groups. In group I, no further manipulations were performed. In groups II and III, collagen hydrogel was injected into the sclerosed vein, respectively, without and seeded with autologous green fluorescent protein-labeled fibroblasts. One week postoperatively, the vein segments were examined for patency and resected for histology. RESULTS: The sclerosed vein segments remained occluded in all animals. Histological examination of luminal thrombi demonstrated numerous viable fibroblasts in group III, whereas there were none in the control specimens from groups I and II. The presence of the injected autologous green fluorescent protein-labeled fibroblasts within thrombi of group III was confirmed by immunohistochemistry. CONCLUSIONS: An injectable tissue-engineered construct enhances sclerotherapy of the jugular vein in a leporine model by reliably delivering fibroblasts that populate the resultant thrombus. Further analysis of this novel therapeutic concept as a means to augment permanent scar formation and reduce luminal recanalization is warranted.

Maintenance of G1 checkpoint controls in telomerase-immortalized endothelial cells.

Here we report the characterization of a series of telomerase-immortalized human umbilical vein endothelial cell lines (i-HUVEC). These cells maintain endothelial characteristics such as marker expression, dependence on basic fibroblast growth factor for proliferation, and the ability to form tube structures on Matrigel. In addition, these cells do not show signs of tumorigenic transformation because their growth is contact-inhibited, serum-dependent, and anchorage-dependent. In addition, i-HUVEC do not grow or survive when implanted subcutaneously in immunocompromised mice. Notably, the i-HUVEC lines maintain normal p53-dependent checkpoint control, inducing expression of p21(Cip1/Waf1) in response to DNA damage. These cells subsequently decrease phosphorylation of pRb and arrest in G1. Furthermore, the i-HUVEC lines maintain normal p53-independent checkpoint control, inducing expression of p27(Kip1) in response to lovastatin treatment, with a subsequent decrease in pRb phosphorylation. Lovastatin-treated i-HUVEC lines undergo a G1 arrest that can be reversed with comparable kinetics to that of low passage HUVEC. Together these data demonstrate that telomerase-immortalized endothelial cells can retain normal phenotypes and cell cycle regulation. This result could have significant implications in the study of angiogenic processes such as tumor growth, wound healing, and the vascularization of engineered tissue.

Binding inhibition of angiogenic factors by heparan sulfate proteoglycans in aqueous humor: potential mechanism for maintenance of an avascular environment.

Aqueous humor is a clear fluid, primarily a blood filtrate, which circulates through the anterior chamber of the eye and bathes the cornea. We explored the possibility that components in the aqueous humor play a direct part in maintaining the avascular environment of the cornea. We report here that heparan sulfate proteoglycan (HSPG) was found in bovine aqueous humor and that it directly inhibits binding of basic fibroblast growth factor and vascular endothelial growth factor to cell-surface heparan sulfate. We demonstrate that this holds true for all heparin binding proteins tested but not for epidermal growth factor, which does not bind heparin. Furthermore, we show, with mathematical modeling, that the concentration of HSPG in aqueous humor (approximately 4 microg/ml), when combined with the clearance of aqueous humor from the eye due to circulation, is sufficient to block the binding of heparin binding growth factors to corneal endothelium. This mechanism suggests a physiological process to control bioavailability of angiogenic growth factors in the cornea.

PPARgamma ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis.

Several drugs approved for a variety of indications have been shown to exhibit antiangiogenic effects. Our study focuses on the PPARgamma ligand rosiglitazone, a compound widely used in the treatment of type 2 diabetes. We demonstrate, for the first time to our knowledge, that PPARgamma is highly expressed in tumor endothelium and is activated by rosiglitazone in cultured endothelial cells. Furthermore, we show that rosiglitazone suppresses primary tumor growth and metastasis by both direct and indirect antiangiogenic effects. Rosiglitazone inhibits bovine capillary endothelial cell but not tumor cell proliferation at low doses in vitro and decreases VEGF production by tumor cells. In our in vivo studies, rosiglitazone suppresses angiogenesis in the chick chorioallantoic membrane, in the avascular cornea, and in a variety of primary tumors. These results suggest that PPARgamma ligands may be useful in treating angiogenic diseases such as cancer by inhibiting angiogenesis.