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Exp Biol Med (Maywood). Author manuscript; available in PMC 2009 April 29. Published in final edited form as: Published online 2008 July 18. doi: 10.3181/0709-RM-251. | PMCID: PMC2674965 NIHMSID: NIHMS106812 |
Ultrastructure of Islet Microcirculation, Pericytes and the Islet Exocrine Interface in the HIP Rat Model of Diabetes Melvin R. Hayden, *†‡1 Poorna R. Karuparthi, *‡ Javad Habibi, *†‡ Guido Lastra, *†‡ Kamlesh Patel, * Chetan Wasekar, *‡ Camila Margarita Manrique, *†‡ Ugur Ozerdem, ¶ Sameer Stas, *†‡ and James R. Sowers *†‡§*Department of Internal Medicine, University of Missouri School of Medicine, Columbia, Missouri 65212 †Department of Endocrinology Diabetes and Metabolism, University of Missouri School of Medicine, Columbia, Missouri 65212 ‡Diabetes and Cardiovascular Research Center, University of Missouri School of Medicine, Columbia, Missouri 65212 §Department of Physiology and Pharmacology, University of Missouri School of Medicine, Columbia, Missouri 65212 Harry S. Truman VA Medical Center, Columbia, Missouri 65201 ¶La Jolla Institute for Molecular Medicine, San Diego, California 92121 Context: The transgenic human islet amyloid polypeptide (HIP) rat model of type 2 diabetes mellitus (T2DM) parallels the functional and structural changes in human islets with T2DM. Objective: The transmission electron microscope (TEM) was utilized to observe the ultrastructural changes in islet microcirculation. Methods: Pancreatic tissue from male Sprague Dawley rats (2, 4, 8, 14 months) were used as controls (SDC) and compared to the 2-, 4-, 8- and 14-month-old HIP rat models. Results: The 2-month-old HIP model demonstrated no islet or microcirculation remodeling changes when compared to the SDC models. The 4-month-old HIP model demonstrated significant pericapillary amyloid deposition and diminution of pericyte foot processes as compared to the SDC models. The 8-month-old model demonstrated extensive islet amyloid deposition associated with pericyte and β-cell apoptosis when compared with SDC. The 14-month-old HIP model demonstrated a marked reduction of β-cells and intra-islet capillaries with near complete replacement of islets by amyloidoses. Increased cellularity in the region of the islet exocrine interface was noted in the 4- to 14-month-old HIP models as compared to SDC. In contrast to intra-islet capillary rarefaction there was noticeable angiogenesis in the islet exocrine interface. Pericytes seemed to be closely associated with collagenosis, intra-islet adipogenesis and angiogenesis in the islet exocrine interface. Conclusion: The above novel findings regarding the microcirculation and pericytes could assist researchers and clinicians in a better morphological understanding of T2DM and lead to new strategies for prevention and treatment of T2DM. Keywords: amylin, angiogenesis, apoptosis, beta cell, islet amyloid, islet fibrosis, exocrine pancreas Type 2 diabetes mellitus (T2DM) has emerged as a pandemic and predictions are that this trend will continue in the future ( 1- 4). Importantly, this pandemic extends beyond the typical middle aged and older aged patient population and now involves our adolescent youth. This alarming trend will place these young patients at risk for more serious complications of end-organ involvement due to a prolonged exposure to the multiple metabolic toxicities associated with these conditions ( 5). Recently, it has been suggested that the islet itself may be an end-organ in T2DM (isletopathy) and further, that the islet may contain an anatomically important region in the peri-islet area termed the islet exocrine interface (IEI) ( 6, 7). T2DM results from pancreatic islet β-cell failure or loss due to apoptosis superimposed on insulin resistance ( 5- 10). The human islet amyloid polypeptide (HIP) rat model of T2DM was created by transfecting the Sprague Dawley control (SDC) rat with the human amylin gene in 2004. The role of the 37 amino acid polypeptide amylin or human amylin derived islet amyloid polypeptide (hIAPP) in the pathogenesis of isletopathy has emerged over the past two decades, and the light microscopic structural abnormalities characterizing this isletopathy have been well described ( 11). Our understanding of the importance of islet amyloid in the pathogenesis of human T2DM has recently increased due to the availability of animal models of T2DM characterized by having amylin derived islet amyloid ( 8- 16). The HIP model is known to spontaneously develop impaired glucose tolerance at 5 months and overt T2DM between the ages of 6 and 10 months of age while consuming a normal rat chow diet ( 11- 13). Recently, the ultrastructural changes of islet amyloid deposition in the 4-, 8- and 14-month-old HIP model have been described ( 17). Transmission electron microscopy (TEM) examination of the islets in this animal model revealed considerable cellular activity and widening at the peri-islet–IEI ( 6, 7). With progressive deposition of islet amyloid this IEI area was characterized by large numbers of capillaries contemporaneous with intra-islet capillary rarefaction due to islet wounding of the vulnerable islet from progressive deposition of amyloid. Therefore, the aim of the current investigation was to evaluate the ultrastructural changes of the microcirculation remodeling with special emphasis on the pluripotent - plastic pericyte ( 6, 7) in the islet of the HIP rat model of T2DM ( Table 1). | Table 1Four Stages of Islet Microcirculation Re-modeling in the HIP Rat Model of Type 2 Diabetes Mellitusa |
Animal Tissue Samples for Transmission Electron Microscopy (TEM) Following harvesting, the tail sections of pancreatic tissue in 2-, 4-, 8- and 14-month-old male SDC and male HIP rat models were thinly sliced and placed immediately in standard TEM fixative. Standard TEM tissue preparation, fixation and staining were employed to study these tissues with TEM as previously described ( 17, 18). Briefly, following secondary fixation, specimens were placed on a rocker overnight, embedded and polymerized at 60°C for 24 hours. 85 nanometer (nm) thin sections were then stained with 5% uranyl acetate and Sato's Triple lead stain for viewing by a Jeol 1200-EX TEM. Light Microscopic Histological Preparation and Staining All pancreatic tissues were harvested from the tail region of the pancreas and immediately fixed in 3% paraformaldehyde, infiltrated and embedded in paraffin. Alpha-Smooth Muscle Actin (α-SMA) α-SMA antibody staining was utilized to specifically identify islet pericytes–myofibroblast-like precursor cells in tissue as described ( 19, 20). Tissue blocks were washed in PBS, suspended in 6.8% sucrose in PBS (pH 7.4), dehydrated in 100% acetone (60 min at 4°C), and embedded in Historesin Plus (3 h at 4°C; no. 7022 2224–861; Leica, Deerfield, IL); or 1 mm3 cubes were washed in PBS, dehydrated in cold ethanol at −4 to −20°C, embedded in Unicryl Resin (British BioCell, Cardiff, UK), and polymerized by ultraviolet light (48 h at −10 to 20°C). 2 μm Historesin Plus sections were stored at 4°C until use; 90-nm Unicryl sections were picked up onto formvar-coated nickel grids and stored at room temperature until use. Platelet Derived Growth Factor Receptor Beta (PDGFR-β) Antibody Staining PDGFR-β antibody has a strong specificity for pericytes and standard fluorescent immunohistochemistry was utilized to identify the presence of pericytes and their islet location in order to supplement the findings of TEM observations. Briefly, 5 μm paraffin sections of 2, 4, 8, and 14 months of HIP pancreas were dewaxed, rehydrated, and antigens were retrieved in citrate buffer at 95°C for 25 minutes. Non-specific binding sites were blocked with 5% bovine serum albumin and 5% normal donkey serum. Sections were then incubated with 1:100 of rabbit polyclonal PDGFR-β antibody in 10 fold diluted blocker. After 24 h, the sections were washed and incubated with 1:300 secondary antibody, Alexa flour-donkey anti rabbit 594, for 4 h. The slides were washed again and incubated with 1:2000 DAPI for 10 minutes. After washing, the slides were mounted with Mowiol and checked under a multi-photon scanning laser confocal microscope (Carl Zeiss, Thornwood, NY). The images were captured by using imaging software (Carl Zeiss, Thornwood, NY). The generation, housing, diet, metabolic collection, sacrifice techniques and the utilization and treatment of animals have been previously described in detail ( 11- 13). The University of California Los Angeles Institutional Animal Care and Use Committee approved all housing, surgical and experimental procedures ( 11, 12). Blood Glucose Values of the Study Animals Whole blood fasting glucose levels were <100, 70, 123, 187 and 213 mg/ml in the SDC (2–14 month), 2-, 4-, 8- and 14-month HIP rat models respectively that were imaged in this TEM study. Detailed weights, metabolic parameters, statistical analyses and light microscopic morphological changes have been previously described in detail regarding this model at ages 2, 5, 10 and 14 months of age ( 11- 13). Weight and insulin levels were unavailable for the HIP rat models imaged in this paper; however, we do know from previous studies that weights increased to approximately 500 grams at age 5 months and remained constant to age 18 months. HIP rat models are known to become insulin deficient between the ages of 10–18 months compared to the SDC models ( 11, 12). The extent of islet amyloid by percentage increased progressively to reach a plateau by age 10 months in the HIP model of T2DM ( 11). Islet amyloid was not present in any of the SDC models because they do not have an amyloidogenic amylin and thus have no islet amyloid deposition for comparison. Unblinded Ultrastructural Evaluation by TEM at 2, 4, 8 and 14 Months 2-Month-Old HIP Model Microcirculation: (Quiescent Stage I) The 2-month-old HIP model was specifically evaluated in this study to be certain there were no early changes in the pericyte–endothelial interactions of the microcirculation. Indeed, the 2-month-old HIP model demonstrated no identifiable islet microcirculation abnormalities by light or TEM compared to its SDC counterparts (Fig. -). Additionally, β-cells were prevalent with abundant insulin secretory granule(s) (ISG) and there was a normal interstitium between intra-islet capillaries and β-cells (50–300 nm) of the 2-month-old HIP model; similar to the SDC models (). In contrast, the peri-islet–islet exocrine interface (IEI) was observed to widen in the 2-month-old HIP model as compared to the SDC 2-month-old model (). This widening of the IEI was associated with the loss of adherens junctions and desmosomes, which normally keep the endocrine and exocrine pancreas in close anatomical proximity in the IEI (). This early widening of the IEI may allow for later remodeling fibrosis and islet amyloid deposition. Additionally, IEI pericytes seemed to be associated with the collagenosis necessary for matrix maintenance of the loose areolar matrix of the IEI and some pericytes were noted to be actively synthesizing collagen at this early age (). 4-Month HIP Model (Islet Wounding Stage II) Islets of the 4-month-old HIP rat model demonstrated substantial pericapillary islet amyloid (hIAPP) deposition as previously reported () ( 17). There was a noticeable loss of pericyte foot processes enveloping the capillary endothelial cells (), and islet amyloid was present between endothelial cells and pericyte foot processes (). Additionally, areas of collagenosis (very early ordered banded-fibrillar collagen) were interspersed with inter-β-cell amyloid deposition, both of which were associated with the pericytes (). The pericapillary islet amyloid resulted in a structural diffusion barrier and interfered with the normal trafficking and docking of ISG of the β-cell and intra-islet capillaries (Fig. , ). In addition to the loss of pericyte foot processes there was a change in the morphology of the cell, in that, pericytes became more cuboidal in shape () as compared to the elongated morphology in the SDC and 2-month-old HIP models (Fig. , ). Importantly, there was noted to be pericyte loss in many intra-islet capillaries (), which could contribute to the capillary rarefaction as the animal model aged. Special Staining with Alpha-Smooth Muscle Actin (α-SMA) and Platelet Derived Growth Factor Receptor-Beta (PDGFR-β) Antibody Staining in the 4-Month-Old HIP Model The 4-month-old HIP model demonstrated the strongest staining signals to α-SMA antibody () when compared to the 2-, 8- and 14-month-old models (results not shown). These observations suggest that the signal for α-SMA identification–activation by antibody staining may precede the migration and/or hyperplasic changes found in the IEI of the 8- and 14-month-old HIP model. In addition to staining pericytes within the peripheral islet and interface regions, the α-SMA antibody stained vascular smooth muscle cells and ductal tissue of the exocrine portion of the pancreas. However, there was no other stromal cell staining noted throughout the pancreas (not shown). This pattern of α-SMA pancreatic staining was also recently noted in the Ren2 model of hypertension and insulin resistance ( 7). Specific pericyte staining with PDGFR-β antibody at this stage demonstrated a tendency for pericyte migration toward the periphery of the islet into the IEI regions (). 8-Month-Old HIP Model (Pericyte and β-Cell Apoptosis Stage III) The 8-month-old HIP rat model demonstrated progressive islet hIAPP deposition as compared to the 4-month-old model and intra-islet capillaries were scarce. In addition to known β-cell apoptosis ( 17) there was novel evidence of pericyte apoptosis (). When found, intra-islet pericytes no longer had their foot processes enveloping the capillary endothelial cells and it appeared as if there was pericyte foot process attenuation and/or loss. The pericyte nucleus and cell body lost it elliptical, elongated shape and assumed a more cuboidal type of morphology in all of the capillaries identified at 8 months (Fig. , ). There was also noted an adaptive remodeling response to the progressive hIAPP replacement remodeling and the peripheral islet cells and matrix at the IEI appeared to physically invade the surrounding exocrine interstitium (). 14-Month HIP Model (Pericyte Differentiation Stage IV) with Near Total Islet Replacement with hIAPP Intra-islet capillaries and functioning β-cells were both extremely difficult to locate in the 14-month-old HIP model due to extensive hIAPP deposition with near total islet replacement. When found buried in a sea of islet amyloid–hIAPP, the β-cells were always accompanied by cells with intracellular lipid droplets and these were assumed initially to be from resident adipocytes ( 17); however, careful evaluation revealed that the lipid droplet formation occurred within pericyte foot processes suggesting differentiation of microvascular pericytes into adipocytes or lipid carrying cells (). Intra-islet collagenosis-fibrosis was observed in the 14-month-old model () interspersed with diffuse islet amyloid deposition. Islet exocrine interface collagenosis for matrix maintenance was observed in the 2-month-old models while orderly fibrillar-banded collagen typical of fibrosis was present in the IEI of the 4-, 8- and 14-month-old models and reminiscent of the early fibrosis found in the Ren2 model of angiotensin II induced hypertension and insulin resistance. Indeed, it is currently thought that the islet pericyte can differentiate into an islet pancreatic stellate–myofibroblast-like cell capable of synthesizing fibrillar-banded collagen interspersed with peri-islet–islet exocrine interface amyloid deposition ( 7). Peri-islet fibrosis is also found to be an important extracellular matrix remodeling change in the Zucker obese model of T2DM and is also a prominent finding in islets of humans with T2DM ( 6, 22, 23). Current observations support the notion that even in the HIP model of T2DM with diffuse amyloid deposition there is concurrent early fibrosis in the intra-islet and IEI regions of the pancreas. Importantly, the 14-month-old HIP model demonstrated capillary gain or angiogenesis in the IEI and a better understanding of the potential role of the pericyte in this anatomical region with abundant capillaries is necessary (Fig. , ). Here the capillary tubes were frequently anuclear () and pericyte foot processes were readily observed in most fields examined (Fig. , ). Curiously, the foot processes adjacent to the capillaries lost their normal finger-like shapes previously observed in controls and the 2-, 4- and 8-month-old models (). Also, there was increased cellularity consisting primarily of pericytes with unusually long foot processes in the islet exocrine interface (Fig. , ). Interestingly, the findings of intra-islet capillary rarefaction and the observation of interface angiogenesis have not been previously described in other animal models of T2DM. In addition to β-cell apoptosis there were also apoptotic changes found in pericytes and their foot processes. When rare intra-islet capillaries were found the pericyte appeared “ghost-like” and less electron dense and this was accompanied by apoptotic changes in the pericyte cytoplasm and foot processes (Fig. , ). Platelet Derived Growth Factor Receptor Beta (PDGFR-β) Antibody Staining PDGFR-β antibody staining was more diffusely present throughout the intra-islet regions in the 2-month-old HIP model; however, the positive staining for pericytes was moderately increased in the 4-month-old model () and markedly increased at the periphery or IEI in the 8- and 14-month model indicating migration and/or hyperplasia in support of the TEM findings (). Since the pericyte is in intimate association with the capillary endothelial cell, these changes are suggestive of intra-islet capillary rarefaction and supportive of islet exocrine interface angiogenesis. The Islet Exocrine Interface (IEI) The IEI may be considered an important structural and functional region, which contains the neurovascular supply including sympathetic and parasympathetic innervations as well as the afferent and efferent vessels (including the insulo-acinar-portal pathway) ( 6, 7). This anatomical region has gained increasing importance during the past decade with the advent of islet transplantation and the need for purification of donor islets ( 24). Also, this region may be important for communication between the endocrine - exocrine pancreatic subdivisions and currently the pancreas may be considered as an integrated organ with two interrelated subdivisions (endocrine-exocrine) in constant communication and functioning synergistically ( 6, 25, 26). Interestingly, the cellular and extracellular matrix remodeling of the IEI cannot be interpreted by light microscopy because the magnification is not sufficient to delineate this anatomical region ( 7). This may be one of the reasons why this specialized anatomical region has been largely overlooked in the past. Imaging studies with TEM has allowed the examination of this specialized anatomical region in greater detail and describe its adaptive–pathological remodeling. As the HIP model aged there seemed to be increased cellular and extracellular matrix activity in this widened and otherwise relatively quiet region. The IEI displayed pericyte hypercellularity in the 4-month-old model () and later manifested an increase in capillaries and pericapillary islet amyloid and collagenosis with early deposition of fibrillar-banded collagen (Fig. , , -). Increased pericytes seemed to be closely associated with the production of new collagen and new capillaries. In some areas of the IEI when there was excessive hIAPP deposition (8- and 14-month models) there seemed to be pericyte foot processes in abundance and some even appeared to be forming capillary tube-like structures (), which has been described in the retina and malignant tumors ( 27). Additionally, the evaluation of the IEI allowed for an improved understanding of how islet cells might extend into the adjacent exocrine tissue noted in the 8-month-old model (). Significant islet amyloid deposition and early fibrosis in this anatomical region occurred concurrently in the HIP model similar to its occurrence in human patients with T2DM ( 6, 23). Importantly, these novel remodeling changes could potentially interfere with paracrine and endocrine communication with the exocrine pancreas and with the afferent delivery of oxygen and nutrients to the islet and the efferent delivery of islet hormones (insulin) and toxicmetabolic byproducts of islet metabolism. These structural changes could have marked detrimental results on islet function and could contribute to the delay of 1st phase insulin secretion and promote dysfunctional islet exocrine interactions. Pericyte Endothelial Interaction in the Microcirculation For a fully competent microvessel to function, pericytes and endothelial cells must function in a synergistic fashion: It takes two (Fig. , ) ( 6, 7). The pericyte is an obligatory mural and mesenchymally derived cell serving many functions to the islet microcirculation including vascular development (post-natal angiogenesis), maturation, remodeling and a stabilizing, supportive-protective role to the capillary endothelial cell (Fig. , ) ( 28, 29). Vascular smooth muscle cells (VSMC) and pericytes share a close homology and it is thought that one may give rise to the other, but there is also evidence that pericytes may arise from native bone marrow progenitor cells ( 29, 30). Circumferential pericyte foot processes are in close contact with capillary endothelial cells and communicate by intimately sharing their basement membranes through readily identifiable ultrastructures termed peg-sockets and adherens junctions () ( 6, 7, 31). Likewise, pericytes may have elongated longitudinal and thin cytoplasmic foot processes, such as those found in the IEI in this investigation (Fig. , ). Pericytes are quite vulnerable to multiple metabolic toxicities and oxidative-redox stress states associated with pre-diabetes and overt T2DM (especially glucotoxicity, dyslipidemia, minimally and advanced glycation endproducts (AGE) and their receptors RAGE) ( 32, 33). For example, pericyte loss in the retinae due to these metabolic abnormalities is commonly observed and may be related to the subsequent development of microaneurysms, acellular capillaries, capillary closure and subsequent capillary loss associated with diabetic retinopathy ( 33, 34). There is considerable phenotypic plasticity of pericytes and they are known to be capable of differentiating into pancreatic stellate cells, fibroblasts–myofibroblasts, chondrocytes, osteoblasts, skeletal myocytes, adipocytes, Leydig cells and the pericyte-VSMC axis ( 7, 35- 37). In addition to the pleiotropic differentiating properties of the pericytes, they, like VSMC seem to be capable of activation–migration ( 7) and frequently undergo apoptosis due to the aforementioned toxicities. The Pericytes' Proposed Role as an Islet Mesenchymal Stem Cell A recent publication supports the notion that mesenchymal stem cells (MSC) reside in virtually all post-natal organs including the endocrine and exocrine pancreas ( 38). These authors point to the possibility that MSC may have a perivascular niche pointing to the pericyte as the possible undifferentiated precursor cell that may be capable of differentiating into adipocytes - lipid carrying cells, pancreatic islet stellate cell–fibroblast–myofibroblasts ( 7) and angiogenic cells associated with adipogenesis, collagenosis and angiogenesis, respectively. Pericyte apoptosis was readily identified as this animal model aged (Fig. , ); however, endothelial cell apoptosis was not identified. Therefore, we hypothesize that the islet pericyte may be more susceptible to injurious islet redox stress stimuli than the endothelial cell similar to the retinal pericyte ( 39). Robust production of injurious islet metabolites associated with T2DM (glucotoxicity, angiotensin II excess, redox–oxidative stress, ROS and islet wounding associated with amyloid deposition) may result in apoptosis; however, less robust production of these islet metabolites might result in pericyte hypertrophy, hyperplasia, increased α-SMA staining and increased matrix metalloproteinase expression. This, in turn, could result in pericyte migration from the intra-islet region or the exocrine pancreas to the IEI while this cell is undergoing differentiation. Further, if the pericyte is destroyed due to apoptosis, its potential as a perivascular MSC could be entirely lost. Interestingly, it was observed that pericytes might be capable of forming closed capillary tubes (). This novel finding is of potential pathophysiological importance since islet amyloid, intra-islet and islet exocrine interface collagenosis – fibrosis and intra-islet adipogenesis is observed at autopsy in human T2DM ( 6, 40). Since the deposition of islet amyloid in this model results in islet wounding it would not be surprising to observe an integral part of the chronic wound healing response: angiogenesis. Thus, we have been able to relate the islet capillary pericyte to islet wounding via a response to injury wound healing mechanism. These observations also suggest that there are remodeling changes associated with early collagenosis-fibrosis, islet adipogenesis and peri-islet–IEI angiogenesis. A potential weakness of this observational study is that only four snapshots in time (2-, 4-, 8- and 14-month-old images) of HIP model microcirculation remodeling and specific pericyte remodeling were conducted. Despite this limitation, these findings have allowed for a significant gain of information regarding the cellular and extracellular islet remodeling as it relates to islet microcirculation and pericyte remodeling changes. The current observational TEM investigation allowed for an examination of the microcirculation within islets and the IEI of the 2-, 4-, 8- and 14-month-old HIP models suggesting 4 stages of islet microcirculation remodeling ( Table 1). Further, this investigation has highlighted the important roles of the IEI and the pluripotent–plastic pericytes involvement in islet microcirculation remodeling. These observations emphasize the impact of both the soluble hIAPP oligomers associated with β-cell apoptosis as well as the impact of the replacement remodeling of the insoluble–fibrillar mature hIAPP with specific attention to the pericyte and critical islet microcirculation. The marked structural remodeling within the islet associated with islet amyloid deposition suggested several pathological processes, including failure of maintaining an intact islet microcirculation involved in the development of T2DM. The novel HIP rat model appears to be an ideal animal model to better understand both the structural and functional changes that occur in human T2DM and better understand the progressive nature of this chronic and progressive pandemic disease. Additionally, these observations have allowed for a more insightful understanding of islet pericyte structural morphology and cellular remodeling as a result of amyloid deposition. In this regard, it is important to note that pericytes constitute a potential mechanism for lifelong repair and regeneration of injured islet tissues. Thus, the possibility of exploiting their regenerative potential is very attractive and could lead to the future development of novel therapies. Collectively this TEM investigation also suggests that islet amyloid deposition and islet pericyte loss may also play an important role in islet transplant failure. Authors wish to acknowledge the Peter C Butler laboratory of the Larry Hillblom Islet Research Center, David Geffen School of Medicine, University of California, Los Angeles, California, who provided tissue specimens for morphologic study with TEM. A special thank you is extended to Tatyana Gurlo, Ph.D. who kindly collected and prepared the TEM tissue for mailing. Additionally, the authors would like to acknowledge the Electron Microscopic Core Center at the University of Missouri–Columbia, Missouri for their excellent help and tissue preparation of animal samples for viewing. This research was supported by NIH (R01 HL73101-01A1), the Veterans Affairs Merit System (0018), and Novartis Pharmaceuticals for James R Sowers, M.D. 1. Amos AF, McCarty DJ, Zimmet P. 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