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Angiogenic capacity of endothelial cells in islets of Langerhans¹

T. LINN², K. SCHNEIDER, H. P. HAMMES^*, K. T. PREISSNER, H. BRANDHORST, E. MORGENSTERN, F. KIEFER and R. G. BRETZEL²

Medical Clinic and Policlinic 3, Justus-Liebig University Giessen, 35392 Giessen;
^* 5th Medical Clinic, Medical Faculty, University of Heidelberg, Mannheim;
Institute for Biochemistry, Faculty of Medicine, Justus-Liebig University Giessen, 35392 Giessen;
Department of Medical Biology, Medical Faculty, University of Saarland, 66421 Homburg/Saar; and
Max-Planck Institute for Vascular Biology, 48149 Münster, Germany

²Correspondence: Medical Clinic and Policlinic 3, Justus-Liebig University Giessen, Rodthohl 6, 35392 Giessen, Germany. E-mail: thomas.linn{at}innere.med.uni-giessen.de

SPECIFIC AIMS

Previous studies suggested that pancreatic islet grafts are exclusively vascularized by the host. We wanted to find out whether isolated islets have the capacity to form endothelial cells in vitro and after being transplanted.

PRINCIPAL FINDINGS

1. Isolated islets contained endothelial cells with proliferative capacity.
   
2. They spontaneously developed branched cords and tube-like structures in fibrin gels (Fig. 1 )
   
3. The cords were composed of endothelial cells
   
4. Formation of cords was stimulated by fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF)
   
5. After islet transplantation, Tie-2 LacZ-positive endothelial cells of both donor(1/3 of total) and recipient (2/3) were identified in the vicinity of or within the graft
   

View larger version (80K): [in this window] [in a new window]   Figure 1. Fibrin gel culture of rat islet in MCDB131-FCS. Micrograph of 2-day-old living culture; newly formed branching microvessels develop from the periphery of the islet. Mean length of all branches gave 318 µm (x100).

CONCLUSIONS AND SIGNIFICANCE

Endothelial cells and/or their progenitors with angiogenic capacity resided within isolated islets of different species and their angiogenic potential was activated by various stimuli. Thus, graft-related endothelia were involved in the process of revascularization after islet transplantation. Endothelial cells could be pivotal in the complex interaction between donor and host in transplantation. It will be important to identify stimulated sources of vascular reorganization in the transplant.

One aim of the present study was to describe endothelial cell outgrowth from isolated islets of Langerhans in different species, including humans. In fact, we found experimental conditions for the protrusion of cells from islets embedded in fibrin into the surrounding matrix. These cells stained positive for von Willebrand factor (vWF). The formation of vWF-positive cords, branches, and tubes occurred from 12 h onward with a maximum after 48 h. No further increase in the formation of these structures was observed when the islets were exposed to the matrix for 72 or 96 h. Although time-dependent extrusion of endothelial cells from cultured collagenase isolated islets was assumed, the occurrence of vWF production provides evidence for their continuing presence in a significant number of islets.

The in vitro islet angiogenesis assay described here offers the opportunity to study mechanisms of the angiogenic cascade under standardized experimental conditions. The distinct advantage of the approach is that ß cells and endothelial cells can be focally delivered into a 3-dimensional matrix, allowing sprouting angiogenesis to occur by invasion into this extracellular matrix. By contrast, other assays lack nonendothelial cells, such as macrophages, which are located within islets of Langerhans and known to contribute markedly to the angiogenic response in vivo.

Growth factors and angiogenesis in isolated islets
As a rule, adult tissue has a low spontaneous rate of angiogenesis. Islets of different species showed significant spontaneous angiogenic activity when subjected to standard culture conditions. Angiogenesis required growth factors and was stimulated by FGF-2, VEGF, and TNF. There is some experimental evidence that the proliferating capacity of islet microvascular endothelial cells is associated with trypsin resistance and increased expression of -1 proteinase inhibitor. This makes them suitable targets for the use of angiogenesis-promoting conditions or substances.

Proper FGF signaling is important for glucose sensing and insulin production of ß cells. FGF-2 is an essential factor for endothelial proliferation of isolated islets.

VEGF added to the culture medium increased the proliferation of islet endothelia. Measurement of O₂ tension in the fibrin matrix resulted in < 3% oxygen so that islets were exposed to hypoxia when embedded into the fibrin matrix. Hypoxia-inducible factor 1 is induced by hypoxia and regulates VEGF signaling and more of a dozen genes encoding glucose transporters and glycolytic enzymes that provide metabolic adaptation to hypoxia. In our model, FGF-2 stimulated islet microvascular endothelial cells to form cords in the absence of TNF-. FGF-2 as well as VEGF promoted substantial formation of cords. The addition of TNF- was able to potentiate VEGF’s effect on endothelial cell proliferation of isolated islets.

Glucose induces islet endothelial proliferation
Intravital microscopic studies demonstrated that the process of vascularization of freely transplanted pancreatic islets is not altered by hyperglycemia and that microvascular perfusion of engrafted islets is elevated even under diabetic conditions. In our experiments glucose increased the length of cords proliferating from the islet into the fibrin matrix. This is concordant with reports demonstrating that hyperglycemia induces a status resembling hypoxia in endothelial cells by intramitochondrial overproduction of reactive oxygen species, activating growth factors, inducing the secretion of vasodilating agents, and increasing vascular leakage. These data were generated in test systems with up to 48 h duration; the influence of long-term hyperglycemia over weeks or months on the fate of cultured or transplanted islets is not known.

Effect of hypoxia and hyperglycemia on transplanted ß cells
Hypoxia has a profound influence on the survival of islet cells and an immediate profound inhibitory effect on insulin secretion. In islets with diameters of 200 µm, centrally located ß cells will be subject to a degree of hypoxia that will have detrimental effects. Insulin content of islet grafts harvested 1 day after transplantation is only 20% of the value before transplantation.

There is some controversy about the influence of hyperglycemia on the long-term fate of transplanted islets. Little is known about the short-term effects of hyperglycemia on the engraftment process. We found that hyperglycemia lasting only a few hours did not inhibit endothelial proliferation but increased the angiogenic activity of isolated islets.

Significant angiogenesis by donor islets
Microcirculation of islets and sequential capillary perfusion plays a pivotal role in glucose homeostasis. Moreover, the microvasculature has been identified as a target site for the graft rejection process. Between days 2 and 4 after transplantation of islets under the skin, the first signs of angiogenesis were observed in mice using intravital microscopy. Capillary sprouts and formation of sinusoidal vessels were supposed to originate from host muscle with feeding vessels penetrating into the islets and breaking into capillaries within the center of the graft. After day 6 red blood cells were observed within the newly formed network.

In some respects our findings are different from this model of islet revascularization. First, there are no comparable studies using kidney or liver, the transplantation sites mostly used for therapeutic transplantation. Second, we found that donor endothelial cells remained in the graft for up to 21 days after transplantation; according to their location at the periphery of the graft surrounded by renal tissue, we hypothesize that they are integrated into new microvessels (Fig. 2 ).

View larger version (106K): [in this window] [in a new window]   Figure 2. Syngenic Islets from Tie-2 LacZ-positive transgenic mice (6–10 wk) transplanted to the kidney capsule of FVB mice. Grafts were removed 5, 10, or 21 days after transplantation (n=5 each per time point). A) LacZ-positive endothelial cell linings (blue) adjacent to a native islet of the mouse pancreas stained for insulin (x250). B) 5 days after Tx, LacZ-positive cells were identified in the connective tissue surrounding and penetrating into the islet graft (x250). C) LacZ-positive spots of endothelial cells in an islet graft 21 days after transplantation (x100). Most spots are located at the border (arrows) of the graft (dark-stained) to the kidney (light color) within the surrounding stroma. Some spots were also found within the graft.

View larger version (25K): [in this window] [in a new window]   Figure 3. This diagram summarizes the findings of the study. Islets isolated from the pancreas with collagenase proliferate to form cords or tubes or even endotheliomas when stimulated by glucose, growth factors, or Pym-T retrovirus, respectively. Transplantation experiments demonstrate that donor and host endothelia meet to form new vessels containing one-third endothelial cells of donor and two-thirds of the recipient origin. This implicates that donor endothelia proliferate and persist within the graft for a longer period than previously assumed.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0615fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0615fje

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