Multiscale and multimodal imaging for three-dimensional vascular and histomorphological organ structure analysis of the pancreas.

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.

Capillary contact points determine beta cell polarity, control secretion and are disrupted in the db/db mouse model of diabetes.

AIMS/HYPOTHESIS: Almost all beta cells contact one capillary and insulin granule fusion is targeted to this region. However, there are reports of beta cells contacting more than one capillary. We therefore set out to determine the proportion of beta cells with multiple contacts and the impact of this on cell structure and function. METHODS: We used pancreatic slices in mice and humans to better maintain cell and islet structure than in isolated islets. Cell structure was assayed using immunofluorescence and 3D confocal microscopy. Live-cell two-photon microscopy was used to map granule fusion events in response to glucose stimulation. RESULTS: We found that 36% and 22% of beta cells in islets from mice and humans, respectively, have separate contact with two capillaries. These contacts establish a distinct form of cell polarity with multiple basal regions. Both capillary contact points are enriched in presynaptic scaffold proteins, and both are a target for insulin granule fusion. Cells with two capillary contact points have a greater capillary contact area and secrete more, with analysis showing that, independent of the number of contact points, increased contact area is correlated with increased granule fusion. Using db/db mice as a model for type 2 diabetes, we observed changes in islet capillary organisation that significantly reduced total islet capillary surface area, and reduced area of capillary contact in single beta cells. CONCLUSIONS/INTERPRETATION: Beta cells that contact two capillaries are a significant subpopulation of beta cells within the islet. They have a distinct form of cell polarity and both contact points are specialised for secretion. The larger capillary contact area of cells with two contact points is correlated with increased secretion. In the db/db mouse, changes in capillary structure impact beta cell capillary contact, implying that this is a new factor contributing to disease progression.

Skeletal Myoblast Cells Enhance the Function of Transplanted Islets in Diabetic Mice.

Islet transplantation (ITx) is an established and safe alternative to pancreas transplantation for type 1 diabetes mellitus (T1DM) patients. However, most ITx recipients lose insulin independence by 3 years after ITx due to early graft loss, such that multiple donors are required to achieve insulin independence. In the present study, we investigated whether skeletal myoblast cells could be beneficial for promoting angiogenesis and maintaining the differentiated phenotypes of islets. In vitro experiments showed that the myoblast cells secreted angiogenesis-related cytokines (vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and stromal-derived factor-1α (SDF-1α)), contributed to maintenance of differentiated islet phenotypes, and enhanced islet cell insulin secretion capacity. To verify these findings in vivo, we transplanted islets alone or with myoblast cells under the kidney capsule of streptozotocin-induced diabetic mice. Compared with islets alone, the group bearing islets with myoblast cells had a significantly lower average blood glucose level. Histological examination revealed that transplants with islets plus myoblast cells were associated with a significantly larger insulin-positive area and significantly higher number of CD31-positive microvessels compared to islets alone. Furthermore, islets cotransplanted with myoblast cells showed JAK-STAT signaling activation. Our results suggest two possible mechanisms underlying enhancement of islet graft function with myoblast cells cotransplantation: "indirect effects" mediated by angiogenesis and "direct effects" of myoblast cells on islets via the JAK-STAT cascade. Overall, these findings suggest that skeletal myoblast cells enhance the function of transplanted islets, implying clinical potential for a novel ITx procedure involving myoblast cells for patients with diabetes.

3D printed VEGF-CPO biomaterial scaffold to promote subcutaneous vascularization and survival of transplanted islets for the treatment of diabetes.

Diabetes is a complex metabolic disease and islet transplantation is a promising approach for the treatment of diabetes. Unfortunately, the transplanted islets at the subcutaneous site are also affected by various adverse factors such as poor vascularization and hypoxia. In this study, we utilize biocompatible copolymers l-lactide and D,l-lactide to manufacture a biomaterial scaffold with a mesh-like structure via 3D printing technology, providing a material foundation for encapsulating pancreatic islet cells. The scaffold maintains the sustained release of vascular endothelial growth factor (VEGF) and a slow release of oxygen from calcium peroxide (CPO), thereby regulating the microenvironment for islet survival. This helps to improve insufficient subcutaneous vascularization and reduce islet death due to hypoxia post-transplantation. By pre-implanting VEGF-CPO scaffolds subcutaneously into diabetic rats, a sufficiently vascularized site is formed, thereby ensuring early survival of transplanted islets. In a word, the VEGF-CPO scaffold shows good biocompatibility both in vitro and in vivo, avoids the adverse effects on the implanted islets, and displays promising clinical transformation prospects.

Efficient Vascular and Neural Engraftment of Stem Cell-Derived Islets.

Pluripotent stem cell-derived islets (SC-islets) have emerged as a new source for ß-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences.

Diabetes as a Pancreatic Microvascular Disease-A Pericytic Perspective.

Diabetes is not only an endocrine but also a vascular disease. Vascular defects are usually seen as consequence of diabetes. However, at the level of the pancreatic islet, vascular alterations have been described before symptom onset. Importantly, the cellular and molecular mechanisms underlying these early vascular defects have not been identified, neither how these could impact the function of islet endocrine cells. In this review, we will discuss the possibility that dysfunction of the mural cells of the microvasculature-known as pericytes-underlies vascular defects observed in islets in pre-symptomatic stages. Pericytes are crucial for vascular homeostasis throughout the body, but their physiological and pathophysiological functions in islets have only recently started to be explored. A previous study had already raised interest in the "microvascular" approach to this disease. With our increased understanding of the crucial role of the islet microvasculature for glucose homeostasis, here we will revisit the vascular aspects of islet function and how their deregulation could contribute to diabetes pathogenesis, focusing in particular on type 1 diabetes (T1D).

The association of islet autoantibodies with the neural retinal thickness and microcirculation in type 1 diabetes mellitus with no clinical evidence of diabetic retinopathy.

OBJECTIVE: To examine the association between islet autoantibodies (IAbs) and the retinal neurovascular changes in type 1 diabetes mellitus (T1DM) with no diabetic retinopathy (NDR). METHODS: This cross-sectional study measured the neural retinal structure and microvascular density of 118 NDR eyes using spectral-domain optical coherence tomography angiography. Retinal structure parameters included retinal thickness (RT), inner retinal thickness (iRT), retina never fibral layer thickness (RNFL thickness), ganglion cell complex thickness (GCC thickness), and loss volume of GCC. Microvascular parameters included vessel density of superficial capillary plexus (sVD), vessel density of deep capillary plexus, and vessel density of choroid capillary plexus. Comparison and correlation analyses of these OCTA parameters were made with various IAbs, including glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase-related islet antigen 2 antibody (IA2A), and zinc transporter 8 antibody (ZnT8A). A general linear model was used to understand the association of IAbs with the retina parameters. RESULTS: The IAb positive (IAbs +) group, which included 85 patients, had thinner RT (235.20 ± 18.10 mm vs. 244.40 ± 19.90 mm at fovea, P = 0.021) and thinner iRT (120.10 ± 9.00 mm vs. 124.70 ± 6.90 mm at parafovea, P = 0.015), compared with the IAb negative (IAbs-) group comprising 33 patients. Furthermore, a more severe reduction of RT was demonstrated in the presence of multiple IAbs. Among the three IAbs, GADA was the most significant independent risk factor of all-round RT decrease (ß = -0.20 vs. -0.27 at fovea and parafovea, respectively, P < 0.05), while titers of IA2A negatively affect sVD in the parafovea (ß = -0.316, P = 0.003). CONCLUSIONS: IAbs are associated with neural retinal thinning and microcirculation reduction in T1DM patients before the clinical onset of diabetic retinopathy.

A microfluidic platform integrating functional vascularized organoids-on-chip.

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics.

Local Dialogues Between the Endocrine and Exocrine Cells in the Pancreas.

For many years, it has been taught in medical textbooks that the endocrine and exocrine parts of the pancreas have separate blood supplies that do not mix. Therefore, they have been studied by different scientific communities, and patients with pancreatic disorders are treated by physicians in different medical disciplines, where endocrine and exocrine function are the focus of endocrinologists and gastroenterologists, respectively. The conventional model that every islet in each pancreatic lobule receives a dedicated arterial blood supply was first proposed in 1932, and it has been inherited to date. Recently, in vivo intravital recording of red blood cell flow in mouse islets as well as in situ structural analysis of 3D pancreatic vasculature from hundreds of islets provided evidence for preferentially integrated pancreatic blood flow in six mammalian species. The majority of islets have no association with the arteriole, and there is bidirectional blood exchange between the two segments. Such vascularization may allow an entire downstream region of islets and acinar cells to be simultaneously exposed to a topologically and temporally specific plasma content, which could underlie an adaptive sensory function as well as common pathogeneses of both portions of the organ in pancreatic diseases, including diabetes.

Impact of Prevascularization on Immunological Environment and Early Engraftment in Subcutaneous Islet Transplantation.

BACKGROUND: The utilization of islet-like cells derived from pluripotent stem cells may resolve the scarcity of islet transplantation donors. The subcutaneous space is a promising transplantation site because of its capacity for graft observation and removal, thereby ensuring safety. To guarantee subcutaneous islet transplantation, physicians should ensure ample blood supply. Numerous methodologies, including prevascularization, have been investigated to augment blood flow, but the optimal approach remains undetermined. METHODS: From C57BL/6 mice, 500 syngeneic islets were transplanted into the prevascularized subcutaneous site of recipient mice by implanting agarose rods with basic fibroblast growth factor at 1 and 2 wk. Before transplantation, the blood glucose levels, cell infiltration, and cytokine levels at the transplant site were evaluated. Furthermore, we examined the impact of the extracellular matrix capsule on graft function and the inflammatory response. RESULTS: Compared with the 1-wk group, the 2-wk group exhibited improved glycemic control, indicating that longer prevascularization enhanced transplant success. Flow cytometry analysis detected immune cells, such as neutrophils and macrophages, in the extracellular matrix capsules, whereas cytometric bead array analysis indicated the release of inflammatory and proinflammatory cytokines. Treatment with antitumor necrosis factor and anti-interleukin-6R antibodies in the 1-wk group improved graft survival, similar to the 2-wk group. CONCLUSIONS: In early prevascularization before subcutaneous transplantation, neutrophil and macrophage accumulation prevented early engraftment owing to inflammatory cytokine production.

Harnessing the benefits of glycine supplementation for improved pancreatic microcirculation in type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is predominantly managed using insulin replacement therapy, however, pancreatic microcirculatory disturbances play a critical role in T1DM pathogenesis, necessitating alternative therapies. This study aimed to investigate the protective effects of glycine supplementation on pancreatic microcirculation in T1DM. Streptozotocin-induced T1DM and glycine-supplemented mice (n = 6 per group) were used alongside control mice. Pancreatic microcirculatory profiles were determined using a laser Doppler blood perfusion monitoring system and wavelet transform spectral analysis. The T1DM group exhibited disorganized pancreatic microcirculatory oscillation. Glycine supplementation significantly restored regular biorhythmic contraction and relaxation, improving blood distribution patterns. Further-more, glycine reversed the lower amplitudes of endothelial oscillators in T1DM mice. Ultrastructural deterioration of islet microvascular endothelial cells (IMECs) and islet microvascular pericytes, including membrane and organelle damage, collagenous fiber proliferation, and reduced edema, was substantially reversed by glycine supplementation. Additionally, glycine supplementation inhibited the production of IL-6, TNF-α, IFN-γ, pro-MMP-9, and VEGF-A in T1DM, with no significant changes in energetic metabolism observed in glycine-supplemented IMECs. A statistically significant decrease in MDA levels accompanied by an increase in SOD levels was also observed with glycine supplementation. Notably, negative correlations emerged between inflammatory cytokines and microhemodynamic profiles. These findings suggest that glycine supplementation may offer a promising therapeutic approach for protecting against pancreatic microcirculatory dysfunction in T1DM.

Immunohistomorphology of pancreatic islet microvasculature and the immunophenotypic analysis of CEPC in adult diabetic rats / Inmunohistomorfología de microvasculatura de islotes pancreatic y el análisis inmunofenotípico de CEPC en ratas diabéticas adultas

SUMMARY: Hyperglycaemia is one of the main causes for the endothelial cell (EC) damage in diabetic patients. Even though circulating endothelial progenitor cells (CEPC) could be used as a prognosis for microvascular complications, there is very little information on the islet microvasculature. We analysed by immunohistochemistry and by flow cytometric immunophenotyping, the expression of CD34 on EC and the expressions of CD31, CD34, CD45 and CD133 on CEPC in Streptozotocin (STZ)-induced diabetic rats. Peripheral blood and tissue specimens were obtained from rats of different treatment regimens: STZ treatment, control saline (NS) and sodium citrate (CB) treatments. Blood cells were exposed to flow cytometric immunophenotyping for CD133, CD31, CD34, CD45 and CD133. While tissues from the pancreas, liver and kidney were routinely processed and stained immunohistochemically for CD34. There was a tendency of an increased in CD45-/CD133+/CD31+/CD34+ cells (0.04 ± 0.11 %) in diabetic rats compared to the controls (CB: 0.03 ± 0.04 %; Saline: 0.01 ± 0.03 %). But there was no significant statistical difference between them. The expression pattern of CD34 on the EC in the organs' vascular beds including arterioles, venules, capillaries and sinusoids was extremely heterogeneous across and within treatment regimens. The ECs in the sinusoids of the liver presented similar CD34 expression patterns across different treatment regimens, while the expression of CD34 on the ECs of sinusoidal capillaries in the pancreas vary with the treatment regimen. We conclude that the degree of endothelial cell damage is not uniform across organs' vascular beds in the rat, contrary to mice and humans. Furthermore, the sinusoids in the pancreas and the kidney may have the same degree of endothelial damage when exposed to the same deleterious causes.

RESUMEN: La hiperglucemia es una de las principales causas del daño de las células endoteliales (EC) en pacientes diabéticos. A pesar de que las células progenitoras endoteliales circulantes (CEPC) podrían utilizarse como pronóstico de las complicaciones microvasculares, hay muy poca información sobre la microvasculatura de los islotes. Se analizaron por inmunohistoquímica y por inmunofenotipificación citométrica de flujo, la expresión de CD34 en EC y las expresiones de CD31, CD34, CD45 y CD133 en CEPC en ratas diabéticas inducidas por estreptozotocina (STZ). Se obtuvieron muestras de sangre y tejidos periféricos a partir de ratas de diferentes regímenes de tratamiento: tratamiento con STZ, solución salina control (NS) y citrato de sodio (CB). Las células sanguíneas fueron expuestas a inmunofenotipado por citometría de flujo para CD133, CD31, CD34, CD45 y CD133. Mientras que los tejidos del páncreas, el hígado y el riñón fueron rutinariamente procesados y teñidos inmunohistoquímicamente para CD34. Se observó una tendencia a un aumento en las células CD45- / CD133 + / CD31 + / CD34 + (0,04 ± 0,11 %) en ratas diabéticas en comparación con los controles (CB: 0,03 ± 0,04 %; Salino: 0,01 ± 0,03 %). Pero no hubo diferencias estadísticamente significativas entre ellos. El patrón de expresión de CD34 en la EC en los lechos vasculares de los órganos incluyendo arteriolas, vénulas, capilares y sinusoides fue extremadamente heterogéneo a través de y dentro de los regímenes de tratamiento. Las EC en los sinusoides del hígado presentaron patrones de expresión de CD34 similares a través de diferentes regímenes de tratamiento, mientras que la expresión de CD34 en las CE de capilares sinusoidales en el páncreas varía con el régimen de tratamiento. Concluimos que el grado de daño de las células endoteliales no es uniforme en los lechos vasculares de los órganos en la rata, en comparación de los ratones y los seres humanos. Además, los sinusoides en el páncreas y el riñón pueden tener el mismo grado de daño endotelial cuando se exponen a las mismas causas deletéreas.

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.