Alfred C. Aplin

Aims/hypothesis: Loss of islet beta cell function and mass are critical in the pathogenesis of type 2 diabetes, a disease in which ~90% of individuals exhibit islet amyloid deposition. Amyloid deposits comprise the normal beta cell secretory product, human islet amyloid polypeptide (hIAPP), the aggregation of which is toxic to beta cells. While the underlying mechanism(s) for toxicity remain unknown, it is likely to involve mitochondrial dysfunction. We have shown that the mitochondrial cholesterol transport protein, steroidogenic acute regulatory protein (StAR), is upregulated in beta cells following amyloid deposition. Here, we examined the role of StAR in the toxicity of islet amyloidosis. Methods: Human islets from non-diabetic donors were cultured under amyloidogenic conditions and StAR expression was examined. StAR expression was also determined in islets isolated from transgenic mice expressing amyloidogenic hIAPP or non-transgenic littermates expressing non-amyloidogenic islet amyloid polypeptide, cultured under amyloidogenic conditions with or without the addition of an amyloid inhibitor. Total islet cholesterol content, mitochondrial cholesterol content, mitochondrial function and cell viability/death were compared in transgenic hIAPP and non-transgenic islets cultured in amyloidogenic conditions. Additionally, StAR localisation to islet cells, as well as its intracellular localisation, was examined. Results: StAR was present in human islets at the mRNA and protein level, and expression increased significantly with amyloid formation in vitro. Further, in hIAPP transgenic mouse islets, StAR expression was amyloid-dependent. StAR was predominantly expressed in beta cells, and the amyloid-induced increase in StAR protein was found specifically in the mitochondrial fraction. While total and mitochondrial cholesterol content was unchanged between non-transgenic and hIAPP transgenic mouse islets cultured under amyloidogenic conditions, increased StAR expression was associated with decreased mitochondrial glucose-stimulated respiration and increased cell death. Conclusions/Interpretation: These findings are consistent with StAR having a pathophysiological role in the beta cell in type 2 diabetes, where its upregulation under conditions of islet amyloid deposition could contribute to mitochondrial dysfunction.

The pancreatic islet vasculature comprises microvascular endothelial cells surrounded by mural cells (pericytes). Both cell types support the islet by providing (1) a conduit for delivery and exchange of nutrients and hormones; (2) paracrine signals and extracellular matrix (ECM) components that support islet development, architecture, and endocrine function; and (3) a barrier against inflammation and immune cell infiltration. In type 2 diabetes, the islet vasculature becomes inflamed, showing loss of endothelial cells, detachment, and/or trans-differentiation of pericytes, vessel dilation, and excessive ECM deposition. While most work to date has focused either on endothelial cells or pericytes in isolation, it is very likely that the interaction between these cell types and disruption of that interaction in diabetes are critically important. In fact, dissociation of pericytes from endothelial cells is an early, key feature of microvascular disease in multiple tissues/disease states. Moreover, in beta-cell replacement therapy, co-transplantation with microvessels versus endothelial cells alone is substantially more effective in improving survival and function of the transplanted cells. Ongoing studies, including characterization of islet vascular cell signatures, will aid in the identification of new therapeutic targets aimed at improving islet function and benefiting people living with all forms of diabetes.

Aims/hypothesis The islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models. Methods Primary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy. Results Aggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (pn=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (pn=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (pn=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (pn=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (ppp<0.001). Conclusions/interpretation Islet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes. Graphical abstract

Progression of atherosclerotic plaques into life-threatening lesions is associated with angiogenesis which contributes to intraplaque hemorrhages and plaque instability. The lack of adequate models for the study of human plaque-induced angiogenesis has limited progress in this field. We describe here a novel ex vivo model which fills this gap. Plaques obtained from 15 patients who underwent endarterectomy procedures were co-cultured in collagen gels with rat aorta rings which served as read-out of human plaque angiogenic activity. The majority of plaque fragments markedly stimulated angiogenic sprouting from the aortic rings while concurrently promoting the outgrowth of resident macrophages from the aortic adventitia. This stimulatory activity correlated with the presence of intraplaque macrophages. Proteomic analysis of plaque secretomes revealed heterogeneity of macrophage-stimulatory cytokine and angiogenic factor production by different plaques. VEGF was identified in some of the plaque secretomes. Antibody-mediated blockade of VEGF had significant but transient inhibitory effect on angiogenesis, which suggested redundancy of plaque-derived angiogenic stimuli. Pharmacologic ablation of adventitial macrophages permanently impaired the angiogenic response of aortic rings to plaque stimuli. Our results show that human plaque-induced angiogenesis can be reproduced ex vivo using rat aortic rings as read-out of plaque angiogenic activity. This model can be used to identify key cellular and molecular mechanisms responsible for the neovascularization of human plaques.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

This chapter describes protocols developed in our laboratory to prepare and analyze angiogenic cultures of rat aorta. Rings of rat aorta cultured in collagen gels produce neovessel outgrowths which reproduce ex vivo key steps of the angiogenic process including endothelial migration, proliferation, proteolytic digestion of the extracellular matrix, capillary tube formation, pericyte recruitment, and vascular regression. The angiogenic response of aortic explants can be stimulated with growth factors or inhibited with anti-angiogenic molecules. Aortic ring cultures can also be used to study tumor angiogenesis. Protocols outlined in this chapter describe how this assay can be modified to investigate the angiogenic activity of cancer cells.

This study was designed to investigate how changes in O₂ levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1–4 % O₂. Additionally, aortic neovessels preformed in atmospheric O₂ lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O₂. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signaling molecules including VEGFR2, NRP1 and Prkc-beta while upregulating expression of VEGFR1. Medium conditioned by hypoxic cultures exhibited angiostatic and anti-VEGF activities likely mediated by sVEGFr1. Hypoxia reduced expression of VEGFR1 and VEGFR2 in endothelial cells while upregulating VEGFR1 in macrophages and VEGF in both macrophages and mural cells. Thus, changes in O₂ levels profoundly affect the endothelial response to angiogenic stimuli. These results suggest that hypoxia-induced angiogenesis is fine-tuned by complex regulatory mechanisms involving not only production of angiogenic factors including VEGF but also differential regulation of VEGFR expression in different cell types and production of inhibitors of VEGF function such as sVEGFR1.

Protocols outlined in this chapter illustrate how to prepare and analyze angiogenic cultures of rat aorta. Aortic rings embedded in gels of extracellular matrix generate vascular outgrowths that can easily be monitored over time with inverted microscopy. The angiogenic response can be measured by counting vessels or with image analysis. Aortic ring cultures can be used to investigate molecular mechanisms of angiogenesis and test the efficacy of stimulators and inhibitors of the angiogenic process. As such this assay is an invaluable tool for both basic and applied angiogenesis research.

The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24 h whereas TLR2, 4, and 8 expression peaks at 24 h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or mural cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial cell types by blocking mural cell recruitment and inducing formation of multinucleated giant cells by macrophages. TLR9-induced mural cell depletion is associated with reduced expression of the mural cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.