Pancreatic Vasculature Research Articles

The pancreatic duct and its arteriovenous relationship: an underutilized aid in the diagnosis and distinction of pancreatic adenocarcinoma from pancreatic intraepithelial neoplasia. A study of 126 pancreatectomy specimens.

Pancreatic intraepithelial neoplasia (PanIN) may be difficult to differentiate from isolated glands of pancreatic adenocarcinoma. We studied the normal relationship between the pancreatic ducts and the pancreatic muscular blood vessels in 126 pancreata, 64 of which were removed for pancreatic adenocarcinoma and 62 for other diagnoses. We also examined the effects that atrophy and PanIN have on this relationship. In normal pancreatic parenchyma and in mild to moderate atrophy, blood vessels are guideposts of the interlobular space. The pancreatic acinar parenchyma ensheathes the pancreatic ductal system and separates it from the muscular pancreatic vasculature. Since these blood vessels do not accompany the pancreatic ducts, the presence of a well-differentiated duct-like structure, which may resemble PanIN, located adjacent to a pancreatic blood vessel should be a clue to the infiltrative and hence malignant nature of that gland. Ducts showing PanIN appear to resist atrophy. Since lobular outlines and vascular landmarks are ultimately lost in severe atrophy, they cannot be relied on to aid in the distinction between PanIN and invasive carcinoma. However, it is unusual to see PanIN1 in such severe atrophy (2%), and although the normal vascular landmarks are no longer there to aid in the interpretation, the presence of well-differentiated duct-like structures resembling PanIN1 in this background should be viewed with suspicion for well-differentiated adenocarcinoma.

Heterogenous expression of nestin in human pancreatic tissue precludes its use as an islet precursor marker.

The discovery of a pancreatic adult stem cell would have significant implications for cell-based replacement therapies for type 1 diabetes mellitus. Nestin, a marker for neural precursor cells, has been suggested as a possible marker for islet progenitor cells. We have characterized the expression and localization of nestin in both the intact human pancreas and clinical human pancreatic islet grafts. Nestin was found to be expressed at different levels in the acinar component of human pancreatic biopsies depending on donor, as well as in ductal structures and islets to some degree. In islets, insulin-producing beta-cells rarely co-expressed the protein, and in the ducts a small percentage (1-2%) of cells co-expressed nestin and cytokeratin 19 (CK19) while most expressed only CK19 (90%) or nestin (5-10%) alone. Assessment of nestin expression in neonatal pancreatic sections revealed an increased number of islet-associated positive cells as compared with adult islets. Nestin immunoreactivity was also found in cells of the pancreatic vasculature and mesenchyme as evidenced by co-localization with smooth muscle actin and vimentin. Samples from post-islet isolation clinical islet grafts revealed a pronounced heterogeneity in the proportion of nestin-positive cells (<1-72%). Co-localization studies in these grafts showed that nestin is not co-expressed in endocrine cells and rarely (<5%) with cytokeratin-positive ductal cells. However, relatively high levels of co-expression were found with acinar cells and cells expressing the mesenchymal marker vimentin. In conclusion we have shown a diffuse and variable expression of nestin in human pancreas that may be due to a number of different processes, including post-mortem tissue remodeling and cellular differentiation. For this reason nestin may not be a suitable marker solely for the identification of endocrine precursor cells in the pancreas.

Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing

In view of the evidence for a role for extracellular ATP in both pancreatic endocrine and exocrine functions, we have investigated the expression of P2X and P2Y receptors in this tissue in neonate and aged rat and mouse. Using immunohistochemistry it was shown that P2X(1), P2X(4), P2X(7), P2Y(1) and P2Y(2) receptors were present in different regions of the rat and mouse pancreas; P2X(3) and P2X(6) receptors were not found, and P2X(5) immunolabelling was only found in some nerves. The pancreatic vasculature of both rat and mouse expressed P2X(1), P2X(2), P2Y(1) and P2Y(2) receptors in the smooth muscle. P2X(1) and P2X(4) receptors were absent in the islets of the neonate pancreas, but were progressively upregulated with age after birth. In contrast, the greatest expression of P2Y(1) in cells from the duct system was in neonate pancreas, while there was no P2Y(1) expression in aged rat pancreas. P2X(7) receptors had a consistent pattern of distribution in all of the groups examined, being located in the outer periphery of the islet. Using antibodies raised against insulin, somatostatin and glucagon, double-labelling immunofluorescence was used to identify P2X(7)-positive cells in different islet of Langerhans cell populations. Our results demonstrated a clear immunoreaction to P2X(7) receptors in islet alpha cells, while no P2X(7) was expressed in beta and delta cells. The significance of the differential expression of P2 receptors in the pancreas during development and ageing, and a possible role for the proliferation and death of the islet cell population are discussed.

Regulated expression of pancreatic renin-angiotensin system in experimental pancreatitis

Our previous studies have provided evidence for the existence of an intrinsic renin-angiotensin system (RAS) in the rat pancreas, which may play a role in the regulation of pancreatic microcirculation and ductal secretion. Such a pancreatic RAS has recently shown to be activated by chronic hypoxia. The activation of a local RAS in the pancreas by chronic hypoxia and its significance of changes may be important for the physiological and pathophysiological aspects of the pancreas. In the present study, the regulation of experimentally induced acute pancreatitis on the expression of local RAS in the pancreas was investigated using Western blot, semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) and immunohistochemical approaches. Results from Western blot demonstrated that experimentally induced pancreatitis caused significantly increased expression of the pancreatic RAS component proteins. In keeping with the protein level, RT–PCR analysis also revealed the enhanced expression of pancreatic RAS genes, notably the angiotensinogen in experimental pancreatitis. Immunohistochemical results further demonstrated that increased immunoreactivity for RAS in experimental pancreatitis was predominantly localized to the endothelia and epithelia of pancreatic vasculature and ductal system respectively. The data indicate that experimental pancreatitis may elicit activation of a local RAS in the pancreas. Such an activation of pancreatic RAS and its significance of differential changes in individual RAS components could play a role in the pathophysiology of acute pancreatitis

Coexpression of receptors for adrenomedullin, calcitonin gene-related peptide, and amylin in pancreatic beta-cells.

Three receptors have been characterized by their ability to bind adrenomedullin (AM): L1, RDC1, and CRLR. Immunohistochemical analysis and RT-PCR showed that all three receptors are expressed by the insulin-producing cells of the islets of Langerhans. RDC1 and CRLR in the presence of particular modifying proteins can also bind calcitonin gene-related peptide (CGRP). Such data suggest that the inhibitory effect caused by both AM and CGRP on insulin secretion is mediated by a direct interaction with the beta-cell. We also identified receptors for amylin, the third member of the AM peptide family, in mouse insulin-secreting cells. The beta-cells located closer to the periphery of the islets had a stronger immunoreactivity for the AM/ CGRP receptors. This observation could be related to a paracrine mechanism, given the proximity of AM- and CGRP-secreting cells (F and delta-cells, respectively), which are located at the periphery of the islets. Interestingly, the smooth muscle cells in the pancreatic vasculature expressed only RDC1, which is in agreement with physiological data showing that AM functions in the cardiovascular system are mainly mediated through a CGRP1 receptor. These data further implicate AM and the other components of its peptide family as important regulators of insulin release.

The tissue renin-angiotensin system in human pancreas.

Evidence exists for the presence of a discrete tissue renin-angiotensin system (RAS) in mouse and rat pancreas that is thought largely to be associated with the vasculature. To investigate this in the human pancreas, and to establish whether the cellular sites of RAS components include the islets of Langerhans, we used immunocytochemistry to localise the expression of angiotensin II (AT1) receptors and (pro)renin, and non-isotopic in situ hybridisation to localise transcription of the (pro)renin gene. Identification of cell types in the islets of Langerhans was achieved using antibodies to glucagon and insulin. The results show the presence of the AT1 receptor and (pro)renin both in the beta cells of the islets of Langerhans, and in endothelial cells of the pancreatic vasculature. Transcription of (pro)renin mRNA, however, was confined to connective tissue surrounding the blood vessels and in reticular fibres within the islets. These findings are similar to those obtained in other tissues, and suggest that renin may be released from its sites of synthesis and taken up by possible cellular sites of action. The results presented here suggest that a tissue RAS may be present in human pancreas and that it may directly affect beta cell function as well as pancreatic blood flow.

Involvement of K+ channel permeability changes in the L‐NAME and indomethacin resistant part of adenosine‐5′‐O‐(2‐thiodiphosphate)‐induced relaxation of pancreatic vascular bed

1. We have previously demonstrated that adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS), a potent P2Y-purinoceptor agonist, relaxed pancreatic vasculature not only through prostacyclin (PGI2) and nitric oxide (NO) release from the endothelium but also through other mechanism(s). In this study, we investigated the effects of an inhibitor of the Na+/K+ pump, of ATP-sensitive K+ (K(ATP)) channels and of small (SK(Ca)) or large (BK(Ca)) conductance Ca2+-activated K+ channels. Experiments were performed at basal tone and during the inhibition of NO synthase and cyclo-oxygenase. 2. In control conditions, ADPbetaS (15 microM) induced an initial transient vasoconstriction followed by a progressive and sustained vasodilatation. In the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 200 microM) the transient vasoconstriction was reversed into a one minute vasodilator effect, which was then followed by a progressive and sustained vasodilatation similar to that observed with ADPbetaS alone. The addition of indomethacin (10 microM) did not significantly modify the profile of ADPbetaS-induced vasodilatation. 3. Ouabain (100 microM) decreased basal pancreatic flow rate and did not modify ADPbetaS-induced relaxation. This inhibitor of the Na+/K+ pump increased the pancreatic vasoconstriction induced by L-NAME or by the co-administration of L-NAME and indomethacin. Ouabain did not modify either the L-NAME or the L-NAME/indomethacin resistant part of the ADPbetaS vasodilatation. 4. The K(ATP) inhibitor tolbutamide (185 microM) did not significantly modify basal pancreatic flow rate and ADPbetaS-induced relaxation. This inhibitor which did not change L-NAME-induced vasoconstriction, significantly diminished the L-NAME resistant part of ADPbetaS-induced vasodilatation. Tolbutamide intensified the vasoconstriction induced by the co-administration of L-NAME and indomethacin. In contrast, the L-NAME/indomethacin resistant part of ADPbetaS vasodilatation was not changed by the closure of K(ATP). 5. The SK(Ca) inhibitor apamin (0.1 microM) did not significantly change pancreatic vascular resistance whatever the experimental conditions (in the absence or in presence of L-NAME or L-NAME/indomethacin). In the presence of L-NAME, the closure of SK(Ca) channels changed the one minute vasodilator effect of ADPbetaS into a potent vasoconstriction and thereafter modified only the beginning of the second part of the L-NAME-resistant part of the ADPbetaS-induced vasodilatation. In contrast, the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation remained unchanged in the presence of apamin. 6. Charybdotoxin (0.2 microM), an inhibitor of BK(Ca), increased pancreatic vascular resistance in the presence of L-NAME/indomethacin. In the presence of L-NAME, the closure of BK(Ca) channels reversed the one minute vasodilator effect of ADPbetaS into a potent vasoconstriction and drastically diminished the sustained vasodilatation. In contrast the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation was not modified by the presence of charybdotoxin. Under L-NAME/indomethacin/charybdotoxin/apamin infusions, ADPbetaS evoked a drastic and transient vasoconstriction reaching a maximum at the second minute, which was followed by a sustained increase in the flow rate throughout the ADPbetaS infusion. The maximal vasodilator effect of ADPbetaS observed was not modified by the addition of apamin. 7. The results suggest that the L-NAME-resistant relaxation induced by ADPbetaS in the pancreatic vascular bed involves activation of BK(Ca), K(ATP) and to a lesser extent of SK(Ca) channels, but the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation is insensitive to the closure of K(ATP), SK(Ca) and BK(Ca) channels.

Angioarchitecture of the atrophic pancreas.

The pancreas has a complex vasculature which comprises both exocrine and endocrine structures. Copper deficiency induces highly selective acinar cell degeneration and progressive noninflammatory lipomatosis in pancreas while Langerhans islets, ducts, and nerves remain unaffected. Pancreatic vasculature was examined in rats that had dietary copper deficiency to characterize changes in the angioarchitecture of the gland. This model was used to assess the degree to which the vasculature of non-acinar components of the gland are potentially altered under conditions of exocrine atrophy. Ultrastructure of pancreas was examined by histology, enzyme histochemistry and immunohistochemistry, corrosion casting and scanning electron microscopy, in situ vascular staining, microsphere injection, biochemical analysis, and morphometry in copper-deficient rats. Results show that no acute angiopathic changes indicative of vascular disorganization accompany atrophy. Only a reduction in the complexity of the capillary beds, which normally vascularize the dense acinar parenchyma, was found. Microsphere quantitation also showed that blood flow to the lipomatous gland remains intact. Furthermore, analysis of the angioarchitecture of the atrophied pancreas supports a largely autonomous blood supply to islets and ducts. These observations support the hypothesis that while the vasculature of the atrophied gland is modified in vascular regions severely targeted by acinar necrosis, the overall structural features of the angioarchitecture are preserved. The atrophied gland thus provides an experimental model to study the vascular routes supplying islet and ductal blood flow within the complex pancreatic circulation.

Angioarchitecture of the atrophic pancreas

The pancreas has a complex vasculature which comprises both exocrine and endocrine structures. Copper deficiency induces highly selective acinar cell degeneration and progressive noninflammatory lipomatosis in pancreas while Langerhans islets, ducts, and nerves remain unaffected. Pancreatic vasculature was examined in rats that had dietary copper deficiency to characterize changes in the angioarchitecture of the gland. This model was used to assess the degree to which the vasculature of non-acinar components of the gland are potentially altered under conditions of exocrine atrophy. Ultrastructure of pancreas was examined by histology, enzyme histochemistry and immunohistochemistry, corrosion casting and scanning electron microscopy, in situ vascular staining, microsphere injection, biochemical analysis, and morphometry in copper-deficient rats. Results show that no acute angiopathic changes indicative of vascular disorganization accompany atrophy. Only a reduction in the complexity of the capillary beds, which normally vascularize the dense acinar parenchyma, was found. Microsphere quantitation also showed that blood flow to the lipomatous gland remains intact. Furthermore, analysis of the angioarchitecture of the atrophied pancreas supports a largely autonomous blood supply to islets and ducts. These observations support the hypothesis that while the vasculature of the atrophied gland is modified in vascular regions severely targeted by acinar necrosis, the overall structural features of the angioarchitecture are preserved. The atrophied gland thus provides an experimental model to study the vascular routes supplying islet and ductal blood flow within the complex pancreatic circulation. Microsc. Res. Tech. 37:520–542, 1997. © 1997 Wiley-Liss, Inc.

Pharmacological characterization of angiotensin II binding sites in the canine pancreas

High affinity 125I-angiotensin II (Ang II) binding sites were characterized in the canine pancreas. Total binding increased with protein concentration and equilibrium was reached within 60–90 min at 22°C. Specific binding was saturable and averaged 70% of total. Scatchard analysis of binding yielded a K D of 0.48±0.18 nM with a B max of 32.8±6.5 fmol/mg protein (mean ± SEM, n = 6). The addition of the reducing agent dithiothreitol increased specific binding two-fold. The rank order of displacement of 125I-Ang II binding by native angiotensin peptides was Ang II ≥ Ang III > AngI > Ang(1–7) > > Ang(1–6) . The use of the specific Ang II antagonists CGP 42112A, PD 123177, and DuP 753 revealed that the pancreas expresses two receptor subtypes. The majority of Ang II binding sites in the pancreas could be classified as type 2 (AT 2), although type 1 (AT 1) sites were also detected. In vitro autoradiography revealed binding sites localized over islet cells, acinar and duct cells, as well as the pancreatic vasculature. In addition, the autoradiographic studies confirmed the predominance of the AT 2 receptor subtype throughout the pancreas.

Progressive changes in pancreatic vasculature accompanying copper deficiency-induced glandular atrophy.

Pancreatic vasculature was studied in rats treated with a dietary copper-deficient regimen to assess and characterize changes in the exocrine angioarchitecture of the gland. The deficient state is known to induce progressive acinar degeneration, atrophy, and subsequent replacement with noninflammatory lipomatosis. This study was carried out since little information exists regarding changes in the vasculature that accompany acute glandular necrosis. Corrosion casts produced from Mercox injected deficient rats were studied using scanning electron microscopy and compared to injected preparations studied by light microscopy. Results show that with initial atrophy of degenerative acini (6-8 wk), pancreatic lobules decreased in size and the volume of the vascular bed reduced. From wk 8 onward, the accumulation of fat cells expanded the configuration of lobules to more normal dimensions. Although vascular interconnectivity within lobules remained reduced, its basic angioarchitecture remained intact and served to vascularize fat cells in lieu of acini. No acute angiopathic changes indicative of vascular disorganization were found. These observations support the hypothesis that the vasculature of the atrophied pancreas, although modified in regions of the gland most severely targeted by acinar necrosis, remains intact and its basic structural features preserved. Preliminary evidence also supports the observation that preservation of the basic elements of lobular angioarchitecture may form the basis for subsequent regeneration of acinar tissue in the reversed deficient state.

Diversion of the gastroduodenal vein: an in situ model of systemic insulin drainage

A technique of diversion of the gastroduodenal vein in a canine model is described to compare long-term metabolic effects of systemic versus portal pancreatic endocrine drainage. The vein was transected at its entrance into the portal vein and either diverted to the inferior vena cava (systemic group) or reanastomosed to the portal vein (portal group). All remaining venous drainage of the pancreas was interrupted. An additional group of animals underwent laparotomy without manipulation of pancreatic vasculature (sham group). Fasting peripheral insulin and glucose values were determined 3 months postoperatively. Fasting insulin values were significantly higher in the systemic group (mean 10.7 ± 1.06 U/ml) than in the portal (5.8 ± 0.70, P = 0.002) and sham (6.4 ± 0.68, P = 0.01) groups. Fasting glucose values were not significantly different in the three groups. At sacrifice, venous thrombosis was noted in one systemically diverted dog (6.7%). All other anastomoses were patent. No significant collateralization was apparent in any group. No significant complications were noted. This procedure simulates the hormonal milieu created by heterotopic pancreatic transplantation while preserving pancreatic innervation and exocrine function, and serves as an excellent model for investigating the effects of systemic hyperinsulinemia on protein, carbohydrate, and lipid metabolism.

High and low molecular weight tracers for the electron microscopical detection of sialoglycoconjugates.

Hydrazide-derivative tracers of different molecular weights have been synthesized for use in the electron microscopical detection of sodium periodate-oxidized sialyl residues of glycoconjugates in various tissues and cells. Haemundecapeptide hydrazide, horseradish peroxidase hydrazide, and Limulus polyphemus haemocyanin hydrazide were obtained by coupling adipic acid dihydrazide to the tracers with the aid of water-soluble carbodiimide. The enzymatic tracers thus prepared retained their peroxidatic activity. On conversion to the hydrazide derivative, the haemocyanin molecule dissociated into its hexameric subunits. In order to test by transmission electron microscopy the ability of the conjugates to bind to the sialoglycoconjugates of endothelial cell surfaces, each tracer was perfused in situ into rat pancreatic vasculature previously oxidized with 1 mM sodium periodate. The three tracers characteristically labelled the various microdomains of the luminal cell coat of the capillary endothelial cell. The electron opacity of the haemocyanin subunits allowed their easy detection when bound to the cell surface or to components of the extracellular matrix. The bound markers were not displaced by a high ionic strength buffer, and did not label desialylated cell surfaces. These results indicate that the three hydrazide-derivative tracers may be useful tools for the electron microscopical detection of cellular and extracellular sialoglycoconjugates.

New perspectives on the microvasculature of the islets of Langerhans in the rat.

The vasculature of the islets of Langerhans was studied in rats using methacrylate corrosion casts and islet reconstructions from stained serial paraffin sections. In corrosion casts, which allowed a three-dimensional view of the pancreatic vasculature, all islets had one or two afferent arterioles, which gave off numerous capillaries to form a glomerular-like network. Islets could be grouped in three classes on the basis of size. Moreover, these classes had preferential locations within the vascular tree: the smaller the islet, the more peripheral. In small islets (those less than 160 micrometers in diameter) efferent capillaries arose from this network and either coalesced at the periphery of the islet or passed through perinsular exocrine tissue before coalescing into venules. However, in intermediate islets (those 160--260 micrometers in diameter) and large islets (those greater than 260 micrometers in diameter) efferent capillaries usually coalesced at the edge of the islet forming an extensive fingerlike network of collecting venules over the islet. This suggested that at least in the rat a large amount of the islet tissue is directly drained by venules. In serial paraffin sections of islets perfused with India ink and stained alternately for B-cells or for non-B-cells, the relation of the blood vessels and the organized array of different cell types making up the islet was discernible. In islets of all sizes, the afferent arterioles entered the islet of all sizes, the afferent arterioles entered the islet at discontinuities of the mantle of non-B-(glucagon, somatostatin, and pancreatic polypeptide) cells. Entering at the B-cell mass, the arterioles broke into capillaries that traversed the B-cell core before passing through the opposite non-B-cell mantle. The afferent capillaries coalesced into collecting venules outside the islet. In intermediate and large islets, the overlying collecting venule network was closely apposed to the mantle. These anatomic findings indicate that in the rat islet only some of the efferent vessels are part of a insuloacinar portal system and that the afferent vessels reach the B-cell core without passing through the non-B-cell islet tissue.

Relationship between development of fibrosis and hemodynamic changes of the pancreas in dogs

Studies were conducted to investigate the relationship between development of fibrosis and hemodynamic changes in the pancreas in dogs. The basal blood flow rate in the atrophied, fibrotic pancreas was preserved at almost the same level as in the normal pancreas. Fibrotic pancreas responded to VIP with increased blood flow, but not to administration of secretin. On the other hand, normal pancreas showed a brisk increase of blood flow in response to both of these agents. Our findings suggest that direct arterial acinar blood flow per gram of acini decreases prominently, whereas direct arterial flow to the islets per gram of islet is well maintained after the development of pancreatic fibrosis. It is also suggested that secretin acts indirectly by stimulating metabolic activity of the acinar cells with a secondary increase in blood flow, whereas VIP has a direct effect on the pancreatic vasculature.

Vascular responsiveness of the in situ perfused dog pancreas

The in situ perfused dog pancreas was utilized to investigate: (a) the contribution of humoral vasoactive agents which are released in shock to maintain the development of pancreatic hypoperfusion, (b) the effectiveness of glucocorticoids as splanchnic vasodilator agents as a possible mechanism of thier beneficial effect in shock, and (c) the pancreatic vascular response to the cardiac glycoside, ouabain. Close intra-arterial injection of norepinephrine, epinephrine, vasopressin and angiotensin in concentrations present in shock animals significantly increased pancreatic vascular resistance, whereas bradykinin, prostaglandins E 1 and E 2 exerted a vasodilator effect. The glucocorticoids, methylprednisolone and dexamethasone, were without net vascular effect in the perfused pancreas at concentrations comparable to those known to be protective in hemorrhagic shock. Ouabain produced a well maintained increase in pancreatic vascular resistance. It is concluded that the release of vasoactive agents may significantly influence the pancreatic vascular response to prolonged hemorrhage. Use of ouabain in the treatment of circulatory shock states could result in severe deleterious effects to the organism because of its prolonged vasoconstriction of the pancreatic vasculature. No evidence of a vasodilator action of glucocorticoids in the pancreatic vasculature was found, suggesting other mechanisms for the protective action of these agents in shock.

PANCREATIC ANGIOGRAPHY, WITH APPLICATION OF SUBSELECTIVE ANGIOGRAPHY OF THE CELIAC OR SUPERIOR MESENTERIC ARTERY TO THE DIAGNOSIS OF CARCINOMA OF THE PANCREAS.

THE diagnosis of pancreatic neoplasms is difficult because of the inaccessibility of the organ to the usual means of examination. Conventional methods of diagnostic, radiography are helpful in only a few cases, usually restricted to those with large tumors that impinge upon or distort adjacent viscera. The x-ray technic of angiographic visualization of blood vessels bordering and situated within the pancreas offers the means of establishing the presence of a tumor of this organ. Angiographic demonstration of the pancreatic vasculature by aortography alone is of no significant value because the finer pancreatic vessels are poorly opacified and obscured by the . . .