Pericytopathy: oxidative stress and impaired cellular longevity in the pancreas and skeletal muscle in metabolic syndrome and type 2 diabetes

Abstract

The pericyte's role has been extensively studied in retinal tissues of diabetic retinopathy; however, little is known regarding its role in such tissues as the pancreas and skeletal muscle. This supportive microvascular mural cell, plays an important and novel role in cellular and extracellular matrix remodeling in the pancreas and skeletal muscle of young rodent models representing the metabolic syndrome and type 2 diabetes mellitus (T2DM). Transmission electron microscopy can be used to evaluate these tissues from young rodent models of insulin resistance and T2DM, including the transgenic Ren2 rat, db/db obese insulin resistant - T2DM mouse, and human islet amyloid polypeptide (HIP) rat model of T2DM. With this method, the earliest pancreatic remodeling change was widening of the islet exocrine interface and pericyte hypercellularity, followed by pericyte differentiation into islet and pancreatic stellate cells with early fibrosis involving the islet exocrine interface and interlobular interstitium. In skeletal muscle there was a unique endothelial capillary connectivity via elongated longitudinal pericyte processes in addition to pericyte to pericyte and pericyte to myocyte cell-cell connections allowing for paracrine communication. Initial pericyte activation due to moderate oxidative stress signaling may be followed by hyperplasia, migration, and differentiation into adult mesenchymal cells. Continued robust oxidative stress may induce pericyte apoptosis and impaired cellular longevity. Circulating antipericyte autoantibodies have recently been characterized, and may provide a screening method to detect those patients who are developing pericyte loss and are at greater risk for the development of complications of T2DM due to pericytopathy and rarefaction. Once detected, these patients may be offered more aggressive treatment strategies such as early pharmacotherapy in addition to life style changes targeted to maintaining pericyte integrity. In conclusion, we have provided a review of current knowledge regarding the pericyte and novel ultrastructural findings regarding its role in metabolic syndrome and T2DM.

Figure 1

Interactions and pathology of pericytes in the pancreas and skeletal muscle. This image depicts the interaction of insulin resistance, free fatty acids—tumor necrosis factor—alpha (TNFα), glucotoxicity and how they individually and synergistically result in ROS generation, along with multiple other metabolic toxicities to develop the activation of protein kinase C and advanced glycation endproducts(AGE)/receptor for AGE/(RAGE) to result in pericyte (Pc) activation, dysfunction and loss (apoptosis). This is followed by endothelial cell (EC) activation, dysfunction, endothelial nitric oxide synthase (eNOS) uncoupling and loss (apoptosis). Ultrastructural remodeling within these beds result in micro macrovascular beds developing leakiness, inflammation, vasoconstriction and a prothrombotic milieu. Each of the microvascular beds presented in this review are detailed including the role of the Pc and EC capillary in the pancreatic and skeletal muscle microcirculation in their respective microvascular beds. Additionally, these changes promote a macrovascular proatherosclerotic milieu and thus, these mechanisms contribute to the development of micro-macrovascular disease in metabolic syndrome and type 2 diabetes mellitus.

Figure 2

It takes two: the pericyte and endothelial cell for the microcirculation. This image demonstrates a large color-enhanced black pericyte (Pc) located superior and structurally related to its adjacent endothelial cell (EC). This slide not only portrays how the two cells are structurally interacting but also depicts pathways that connect Pcs and ECs in normal states of health and also those involved in disease states from our past studies. These include the necessary roles of redox stress with the Pc, which is believed to be more sensitive to oxidative redox stress (*) as compared to ECs. We have noted that the Pcs undergo apoptotic changes prior to EC loss and capillary rarefaction. We have noted that only the alpha smooth muscle actin is upregulated in the pericyte indicating activation as well as platelet-derived growth factor beta receptor (PDGFβ R). Additionally, only the Pc is capable of synthesizing vascular endothelial cell growth factor (VEGF) and that the EC have only the VE GF Receptors-1–fms-like tyrosine kinase-1(flt-1) and VE GFR-2–kinase insert domain-containing receptor (KDR). Importantly, only the ECs are capable of synthesizing PDGFβ and only the Pcs have PDGFβ R. Insert depicts the Pc–EC peg-socket type of ultrastructural cell-cell interaction.

Figure 3

Pericyte-endothelial morphology and connections. (A) demonstrates an intraislet circumferential pericyte (Pc) surrounding an endothelial cell (EC) with its cytoplasmic pericyte processes (PcP). Note that the PcP come in intimate contact with the EC at specific sites termed peg sockets (PS) and adherens junctions (Aj) (arrows). Also note the loose areolar interstitium (int) surrounding these two cells. These contact points are demonstrated in greater detail in (B–D). Magnification x15,000. Bar = 500 nm. (B) isan exploded image of (A) and the Pc has been darkly highlighted to better depict the communication contact structures between the Pc and EC. (C) illustrates both types of endothelial-pericyte communication, cell-cell connections. Note the Peg socket connection (arrowhead) and the adherens junction (arrows) between the Pc and the EC. These structures provide direct communication between these two cells via specific connections containing connexin 43 (Cx 43). Magnification x50,000. Bar = 100 nm. (D) depicts the peg socket connection between the Pc and EC at higher magnification and further demonstrates the presence of a caveolae (arrows), which also provides communication between these two cells. Magnification x150,000. Bar = 50 nm. Insert (d) is an exploded image of the adherens junction in (C). Original magnification x50,000.

Figure 4

Pericyte capillary connectivity in the islet exocrine interface and exocrine pancreas. This image depicts the pericyte (Pc) connectivity of capillaries (asterisks) via the long pericyte processes (PcP) (arrows) within the endoacinar matrix of the exocrine pancreas and islet exocrine interface (IEI ) (arrowhead). The IEI between the islet and the exocrine tissue within the pancreas is continuous as depicted in insert (a). Within exploded insert (a) note the pericyte processes (PcP arrows) traversing the IEI (arrowhead) and the endoacinar matrix demonstrating that these two matrices are continuous within the normal pancreas. This continuous matrix is important as it will later become totally fibrosed and there has been demonstrated a loss of matrix communication due to fibrosis in humans and models with the metabolic syndrome and type 2 diabetes mellitus [16]. Insert (b) is a minimized—inverted image, which demonstrates the IEI and endoacinar matrix in black. This image is reminiscent of a roadmap and that is why we have termed the IEI and endoacinar matrix the “Pancreatic Superhighway.” Original magnification X250. Bar = 5 μm. Modified and adapted with permission [16].

Figure 5

Capillary connectivity via long cytoplasmic pericyte processes in soleus muscle. (A) demonstrates microvascular capillaries (asterisks) in the endomysium between two soleus myocytes in the Sprague Dawley control model (SDC). Note the long cytoplasmic pericyte processes (pp) (arrows) connecting the capillaries. Magnification x3,380. Bar = 40 μm. (B) depicts the endomysial pericyte processes at higher magnification connecting two capillaries (asterisks) via long pericyte processes (pp) (arrows) between two soleus myocytes. Magnification x8,216. Bar = 10 μm. (C) demonstrates two capillaries (asterisks) connected via pericyte processes (pp) (arrows) within the endomysium. Note the mitochondrial mound (Mt-M) and the intermyofibrillar mitochondria (Mt) in the soleus muscle. Also note how the capillaries(asterisk) seems to be embedded within lacunar-shaped structures in the soleus myocyte. Magnification x10,003. Bar = 10 μm. (D) depicts a pericyte process (pp) within the endomysium of the Ren2 model extending close to the soleus myocyte. Note the pp secretory vesicles (SV) lined up in a row enabling direct communication with the soleus myocyte shown at higher magnification in insert (d) (arrows). Insert (d′) further portrays a caveolae of the pp. The soleus sarcolemma also depicts receptive caveoli arrowheads within the myocyte allowing for direct communication between the pp and the myocyte within the endomysium. These organelles may allow for direct paracrine communication between pericyte processes and the myocytes. Magnification x50,000. Bar = 1 μm.

Figure 6

Pericyte-endothelial cell—pericyte-pericyte connections in pancreas and skeletal muscle of rodent animal models with cardiometabolic syndrome and type 2 diabetes mellitus. (A) depicts the pericyte (Pc) endothelial capillary connection in the gastrocnemius muscle of the Ren2 animal model. This connection appears similar to foot plate connections in neural tissue and insert portrays an exploded view of this pericyte process (pp) connection. Note the marked redundancy of the endoplasmic reticulum (ER ). Magnification x2,500. Bar = 0.05 μm. (B) demonstrates the interaction between the Pc and the EC surrounding the capillary lumen (CL) within the endoacinar matrix of the db/db pancreas. Note cytoplasmic pericyte processes (arrows) within the endoacinar matrix. Magnification X600. Bar = 2 μm. (C) portrays the cytoplasmic pericyte processes of one pericyte (Pc-1) connecting and communicating with another pericyte (Pc-2) (arrows) in the islet exocrine interface (IEI ) of the db/dbWT control mouse model pancreas. Note the presence of secretory vesicles within the Pc-1cytoplasmic process (arrowhead). Magnification x4,000. Bar = 0.5 μm. (D) demonstrates Pc-Pc connection of one pericyte (Pc-1) to another pericyte (Pc-2) in the Ren2 model. Inserts 1–3 depicts the marked elongation of the pp, which ranges from 40–60 μm. Magnification X1,200. Bar = 2 μm. Insert 4 demonstrates a higher magnification image of the connection between Pc-1 and Pc-2 via the cytoplasmic pericyte process (pp). Magnification x4,000. Bar = 0.5 μm.

Figure 7

marked fibrosis of end-stage pancreatic failure in a 58 y/o female patient with type 2 diabetes mellitus (T2DM). This photoshop enhanced Verhoeff's Van Gieson (VV G) stained image demonstrates the marked fibrosis in all of the matrices of the pancreas i.e., intra-islet, islet exocrine interface, endoacinar, interlobular, perivascular and periductal matrix by the crimson red staining of collagen. Also note the arteriosclerotic changes and adipose deposition in conjunction with acinar loss and atrophy. This extensive fibrosis of the Pancreatic Superhighway matrix (Fig. 3) results in the loss of communication between the endocrine and exocrine pancreas in T2DM. Magnification x100. Bar = 200 μm. Artery = A, vein = V, duct = D and islet exocrine interface = IEI. Insert depicts the presence of intra-islet and islet exocrine interface amyloid deposition, which is known to occur concurrently with fibrosis in humans with the metabolic syndrome and T2DM. Micrograph stained with Congo-Red and viewed with crossed polarized light, which allows the gold colored islet amyloid to be detected. Original magnification x400. (B) demonstrates at higher magnification, the boxed in area from (A). Note the crimson red staining of collagen in the regions of the islet exocrine interface (IEI ), the interlobular interstitium (ILI) and the interacinar interstitium (IAI). Magnification x400. Bar = 200 μm. (C) depicts collagen deposition–fibrosis within the islets and the peri-islet–islet exocrine interface of the human pancreas utilizing picrosirius red staining and bright field microscopy. Magnification X400. Insert demonstrates this same islet in crossed polarized light and note how types I and III collagen stain a golden color with crossed polarized light. Minimized insert also magnified x400; bar = 200 μm. Images in (A–C) demonstrate the end-stage fibrosis of pancreatic failure found in metabolic syndrome and T2DM. (A–C) modified with permission [16]. (D) illustrates the early collagen deposition of fibrosis found in the young Ren2 model. The perivascular, periductal (asterisks) and interlobular fibrosis stained yellowish gold (arrows) appears to occur prior to and more robust than peri-islet–islet exocrine interface fibrosis. Magnification x100. Bar = 200 μm. In contrast, the insert of an exploded view demonstrates the lack of peri-islet–islet exocrine interface fibrosis (arrows) as compared to the interlobular perivascular and periductal fibrosis. Original magnification x100 and exploded. There appears to be a temporal and spatial deposition of collagen in this early fibrosis found in the Ren2 model. Images adapted with permission [20].

Figure 8

Widening and hypercellularity of the islet exocrine interface. (A) demonstrates the 40-fold widening of the islet exocrine interface (IEI ) found in the young db/db model of type 2 diabetes mellitus (T2DM) as compared to its lean wild-type non-diabetic littermate aged 7 weeks (insert). Note the four pericytes (Pc) within the widened IEI. Magnification x500. Bar = 2 μm. (B) depicts the IEI hypercellularity with multiple Pc in the young (9-week-old) Ren2 model. IC, islet cells; Pc, pericyte; RBC, red blood cell; ZG, zymogen granules. Magnification x2,500. Bar = 2 μm. (C) portrays pericyte (Pc) and monocyte (M) infiltration of the IEI in the 4-month-old HIP model with insert demonstrating widening of the IEI (insert) similar to the db/db model and the Ren2 model in (A and B). Each of the three animal models studied demonstrate a widening and infiltration of Pc and inflammatory monocytes. Magnification x2,500. Bar = 2 μm. Insert magnification x1,500. Bar = 1 μm. (D) depicts specific inflammatory monocyte infiltration (arrows) within the IEI of the 7-week-old db/db model of T2DM. There were no inflammatory cells found in any of the control models examined (db/dbWT lean littermate controls). Magnification x1,500. Bar = 1 μm in each plate. Modified with permission [16].

Figure 9

Pericyte degeneration in the islet exocrine interface, intraislet and endomysium of Ren2 soleus skeletal muscle. (A) depicts a normal electron dense pericyte (Pc) in the widened islet exocrine interface (IEI ) (dashed double arrow) of the db/db model of type 2 diabetes mellitus (T2DM). CL, capillary lumen; EC, endothelial cell; IC, islet cell. Magnification X1,500. Bar = 2 μm. (B), in contrast to (A), demonstrates a proapoptotic Pc ghost cell in the widened IEI (dashed double arrow) from the same db/db model of T2DM. Note that this Pc is much less electron dense with less visible—less electron dense cytoplasmic organelles such as the endoplasmic reticulum. Magnification x800. Bar = 2 μm. (C) demonstrates the close relationship of an apoptotic Pc to an islet capillary with intracellular apoptotic bodies (arrows) and degeneration of cytoplasmic organelles surrounded by islet amyloid (*) in the 8-month-old human islet amyloid polypeptide (HIP) rat model prior to the development of islet capillary rarefaction. Magnification x3,000. Bar = 2 μm. (D) depicts an apoptotic Pc in the endomysial region of the soleus skeletal muscle of the Ren2 model at 9 weeks of age. Note the loss of cytoplasmic organelles and the decrease in electron density. Exploded insert depicts the cytosolic apoptotic loops (arrows) characteristic of apoptosis. Note how the microcirculatory capillary is nested within a lacunae of the soleus muscle. Endomysium, white; Pc, green; capillary lumen (CL), yellow; endothelium, red; myocyte nucleus (N), blue; mitochondria (Mt), purple; soleus skeletal muscle, rust in color. X marks a region of mitochondrial degeneration in the mitochondrial mounding of the soleus skeletal muscle. Magnification x800. Bar = 2 μm.

Figure 10

Progressive islet amyloid deposition in the aging hip rat model. (A–D) depict the progressive deposition of human islet amyloid within the islets of transfected Sprague Dawley rat with human amyloidogenic amylin, utilizing the Verhoeff van Gieson (VV G) Stain. Note the early pericapillary diffusion barrier created as human islet amyloid encompasses the intraislet capillaries (X) and deposits between the more centrally located β-cells of the islet (asterisk) in (B). Also note the progressive deposition of islet amyloid in the eight- and 14-month-old models in (C and D).

Figure 11

The skeletal muscle trinity: pericyte, mitochondria and capillary loss: rarefaction. This Photoshop-colorized image of the Ren2 gastrocnemius skeletal muscle demonstrates three crucial losses (trinity) involved in capillary rarefaction found in both the islet and skeletal muscles of patients and animal models with metabolic syndrome and type 2 diabetes mellitus. Number 1, (first) to be lost is the (green) pericyte (Pc), Number 2 (second) the subsarcolemmal and perinuclear mitochondria (purple) and number 3, the (third) to be lost the capillary with yellow capillary lumen (CL) and red blood cell (red) representing capillary rarefaction within the endomysial interstitial matrix (dashed arrows) of the gastrocnemius skeletal muscle (rust). These losses may contribute significantly to the development of skeletal muscle insulin resistance, which are known to be important in the compensatory hyperinsulinemia by the beta-cells of the islet. These losses may contribute to the development of impaired glucose tolerance and overt type 2 diabetes mellitus when the compensatory hyperinsulinemia can no longer be sustained due to beta cell secretory deficit or loss. Also note the green pericyte process (PcP) (arrows). These similar changes are also likely to be manifest within the pancreatic islet (Fig. 7C) prior to islet rarefaction. Magnification x5,000. Bar = 1 μm.