William P. Duguid

Expression of Endothelin-1 in the Lungs of Patients with Pulmonary Hypertension

BACKGROUND Pulmonary hypertension is characterized by an increase in vascular tone or an abnormal proliferation of muscle cells in the walls of small pulmonary arteries. Endothelin-1 is a potent endothelium-derived vasoconstrictor peptide with important mitogenic properties. It has therefore been suggested that endothelin-1 may contribute to increases in pulmonary arterial tone or smooth-muscle proliferation in patients with pulmonary hypertension. We studied the sites and magnitude of endothelin-1 production in the lungs of patients with various causes of pulmonary hypertension. METHODS We studied the distribution of endothelin-1-like immunoreactivity (by immunocytochemical analysis) and endothelin-1 messenger RNA (by in situ hybridization) in lung specimens from 15 control subjects, 11 patients with plexogenic pulmonary arteriopathy (grades 4 through 6), and 17 patients with secondary pulmonary hypertension and pulmonary arteriopathy of grades 1 through 3. RESULTS In the controls, endothelin-1-like immunoreactivity was rarely seen in vascular endothelial cells. In the patients with pulmonary hypertension, endothelin-1-like immunoreactivity was abundant, predominantly in endothelial cells of pulmonary arteries with medial thickening and intimal fibrosis. Likewise, endothelin-1 messenger RNA was increased in the patients with pulmonary hypertension and was expressed primarily at sites of endothelin-1-like immunoreactivity. There was a strong correlation between the intensity of endothelin-1-like immunoreactivity and pulmonary vascular resistance in the patients with plexogenic pulmonary arteriopathy, but not in those with secondary pulmonary hypertension. CONCLUSIONS Pulmonary hypertension is associated with the increased expression of endothelin-1 in vascular endothelial cells, suggesting that the local production of endothelin-1 may contribute to the vascular abnormalities associated with this disorder.

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (INGAP) is a product of a novel gene expressed in regenerating hamster pancreas. Northern blot analysis showed a strong single transcript of 850 bp at 1 and 2 d after CW that disappeared by the 6th day and was absent from untreated control pancreata. INGAP gene is expressed in acinar cells, but not in islets. Western blot analysis demonstrated the presence of INGAP in Ilotropin but not in extracts from control pancreata. A synthetic pentadecapeptide, corresponding to a region unique to INGAP, stimulated a 2.4-fold increase in [3H]thymidine incorporation into hamster duct epithelium in primary culture and a rat pancreatic duct cell line but had no effect on a hamster insulinoma tumor cell line. A portion of human INGAP gene was cloned and appears to be highly homologous to the hamster gene. This data suggests that the INGAP gene is a novel pancreatic gene expressed during islet neogenesis whose protein product is a constituent of Ilotropin and is capable of initiating duct cell proliferation, a prerequisite for islet neogenesis.

Cell loss in isolated human islets occurs by apoptosis.

Purified islet allografts have largely failed to maintain long-term glucose homeostasis in human recipients, and the reasons for this are unclear. It is noteworthy, however, that islet isolation destroys or removes cellular and noncellular elements of the pancreas that could play an important role in supporting islet survival. The purpose of this study was to determine whether human islet isolation leads to the induction of programmed cell death. Human islets were enzymatically isolated from cadaveric donor pancreata using Liberase or Collagenase P, purified over a discontinuous BSA gradient, then cultured in RPMI 1640 at 37°C in 5% CO2 for ≤7 days. Islets were examined daily by routine histology and immunocytochemistry for islet hormones, DNA fragmentation [cell death; enzyme-linked immunosorbent assay (ELISA) and TUNEL assay] and for transglutaminase (TG) activity, two indicators of apoptosis. TG activity and DNA fragmentation increased by 1,000% and 1,890%, respectively (p < 0.05) This corresponded to the appearance of pyknotic nuclei on light microscopy, the presence of apoptotic bodies on electron microscopy, and the demonstration of TUNEL-positive cells. These were present primarily in a distribution that corresponded to the insulin-immunoreactive cells. At 5 days, 31.4 ± 2.2% of islet cells were TUNEL positive. In summary, apoptosis of islet cells appears soon after islet isolation, and involves primarily the &bgr; cell. This is the first report of apoptosis of islet cells after human islet isolation. The loss of &bgr;-cell mass could be implicated in the failure of islet transplantation and merits further investigation.

A new approach to the induction of duct epithelial hyperplasia and nesidioblastosis by cellophane wrapping of the hamster pancreas

There is ample evidence that a relationship exists between duct epithelial hyperplasia and carcinoma of the pancreas. However, no experimental system exists to investigate the mechanisms involved. A new model is described in the Syrian golden hamster for inducing duct epithelial hyperplasia and nesidioblastosis. The head of the pancreas is wrapped with cellophane tape; ligation of the duct is not involved and there is no evidence of diffuse pancreatitis. Preliminary studies have revealed that this model results in periductal fibrosis in relation to the cellophane thus producing partial duct obstruction. Concomitant with the fibrosis there is duct epithelial hyperplasia in the head of the gland, while peripherally there is ductular proliferation and the initiation of nesidioblastosis. It is believed that this model will be of use in studies of pancreatic carcinogenesis and will thus enhance our knowledge of the interrelationships between etiologic factors, precursor lesions, and pancreatic cancer. This is of particular importance in the early recognition of this neoplasm in man.

Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s)

SummaryPartial pancreatic duct obstruction in the hamster leads to the induction of endocrine-cell differentiation and new islet formation. We prepared cytosolic extracts from the partially obstructed pancreas and identified one, which when administered i.p., produced significant increases in the incorporation of tritiated thymidine by ductular and islet cells, as well as a corresponding increase in islet mass. In this study, we evaluate the ability of this extract to reverse streptozotocin diabetes mellitus. Hamsters were treated i. p. twice daily for 7 weeks with either 0.9% NaCl (saline) (n=10) or a cytosol extract (n=10) prepared previously from partially obstructed hamster pancreata. All animals in the cytosol group survived vs only 60% of the saline group (p=0.02). Random blood glucose levels were greater than 22.2 mmol/l in 90% of the saline group vs 40% in the cytosol group (p<0.05). Pancreatic tissue from the surviving saline animals and from persistently hyperglycaemic cytosol-treated animals, showed intra-cytoplasmic vacuolation of islet cells, a characteristic lesion of sustained hyperglycaemic states. Vacuolation was not observed in normoglycaemic extract treated animals. Islets in hyperglycaemic animals demonstrated a profound decrease or absence of immunoreactive insulin, compared to an abundance of immunoreactive beta cells in cytosol-treated animals that reverted to normoglycaemia. In this group, single cells or nests of cells stained for insulin or glucagon cells were identified in ductal epithelium in association with cells budding from the duct. Morphometric analysis of pancreata in reverted cytosol-treated animals showed a new population of small islets compared with saline controls and an increased islet mass. In summary, streptozotocin diabetes can be reversed by new islet formation induced by local pancreatic growth factors, the exact nature of which remains to be determined.

The effect of cellophane wrapping of the pancreas in the Syrian golden hamster: autoradiographic observations.

We examined the effects of cellophane wrapping of the pancreas on the age-related uptake of tritiated thymidine (3H-TdR) by the differentiated cell types of the pancreas of the Syrian golden hamster. Fifty-two hamsters were studied. At 7 weeks of age, hamsters underwent cellophane wrapping (n = 32) or were allocated to a control group (n = 20). Animals 8–22 weeks of age (four at each interval) received 3H-TdR (2 μCi/g) intraperitoneally and were killed 1 h later. Pancreatic tissues from each animal was processed for autoradiography. The percent of acinar cells labeled with 3H-TdR at 8 weeks, in control and wrapped animals, was 1.17 ± 0.26 and 1.51 ± 0.38 respectively (p = N.S.). In control animals, this steadily diminished to 0.02 ± 0.00 at 22 weeks. In wrapped animals, there was less of a tendency for acinar cell labeling to decrease with age, and the percent of labeled acinar cells in wrapped animals at 22 weeks was 0.05 ± 0.00. The percent of ductular cells labeled with 3H-TdR at 8 weeks in control and wrapped animals was 0.24 ± 0.24 and 0.98 ± 0.24, respectively (p = N.S.), and at 22 weeks was 0.13 ± 0.90 and 0.60 ± 0.03, respectively (p < 0.01). The percent of islet cells labeled with 3H-TdR at 8 weeks in control and wrapped animals was 0.16 ± 0.01 and 0.42 ± 0.01, respectively (p < 0.05), and at 22 weeks was 0.18 ± 0.01 and 0.63 ± 0.05 (p < 0.05), respectively. In the major ducts in the head of the gland, the number of epithelial cells per millimeter duct length that were labeled with 3H-TdR in control animals was stable throughout the study: 3.25 ± 1.5 at 8 weeks and 3.35 ± 1.3 at 22 weeks. In the wrapped group, the number of labeled duct epithelial cells was greatest at 8 weeks (22.5 ± 7.5) and then declined to 13.7 ± 4.5 at 12 weeks, at which time it stabilized. It is concluded that cellophane wrapping of the pancreas is a mitogenic stimulus that affects all cell types in the pancreas of the Syrian golden hamster. The greatest trophic affect was on cellular elements of the ductal system and on islet cells.

Pancreatic schwannoma: report of two cases and review of the literature.

Solitary intrapancreatic schwannoma is a rare tumor. We present two patients with this tumor and review 13 previously reported cases from the English-language literature. While the final diagnosis was made based on pathological examination of the tumors, both computed tomography scan and magnetic resonance imaging helped establish the benign nature of the lesion, narrow the differential diagnosis, and define the anatomical locations of the small tumors. Both tumors were treated by enucleation from the surrounding pancreatic parenchyma, and both patients, after 2 years of follow-up, are alive and well. It is concluded that multimodality radiologic investigations are useful in the workup of unusual pancreatic masses. In addition, based on the known biologic behavior of schwannomas occurring elsewhere in the body, simple enucleation, rather than more radical resection, is likely to be adequate therapy for these tumors.

Expression of endothelin-1 in pancreatic tissue of patients with chronic pancreatitis.

Chronic pancreatitis is characterized by the presence of an inflammatory infiltrate, progressive destruction of acinar cells, and fibrosis. The finding that endothelin‐1, an endothelium‐derived peptide with vasoconstrictive and mitogenic properties, reduces pancreatic blood flow in normal rats suggested that the peptide may be associated with the reduced pancreatic flow seen in animal models of chronic pancreatitis and in the morphological abnormalities of the disease. The aim of this study was to investigate sites of endothelin‐1 expression in the pancreas of normal subjects and patients with chronic pancreatitis. The techniques of immunohistochemistry, in situ hybridization, and Northern blotting were used. Endothelin‐1‐like immunoreactivity was localized predominantly to islet cells both in normal subjects and in patients with chronic pancreatitis. Semi‐quantitative analyses of immunostaining showed that endothelin‐1‐like immunoreactivity in islet cells of patients with chronic pancreatitis was greater than in normal subjects. Co‐localization studies with glucagon, insulin, somatostatin, and pancreatic polypeptide showed that endothelin‐1‐like immunoreactivity co‐exists with glucagon and insulin. There was no apparent co‐existence of endothelin‐1‐like immunoreactivity with somatostatin or pancreatic polypeptide. Endothelin‐1 mRNA was expressed in sites similar to those of the immunostaining, as well as in vascular endothelial cells. Northern blot analysis showed an increase in the expression of endothelin‐1 mRNA in the patient population. There was a significant correlation between intensity of endothelin‐1 immunostaining and severity of fibrosis in the patients with chronic pancreatitis. These findings suggest that an elevation in local expression of endothelin‐1 may be associated with the morphological and haemodynamic changes of chronic pancreatitis.

Induction of Islet Cell Differentiation and New Islet Formation in the Hamster-Further Support for a Ductular Origin

Partial obstruction of the adult hamster pancreas leads to islet cell differentiation and new islet formation. From morphologic and morphometric observations, we have tentatively identified the source of the new islet tissue to be from cells in the ducts. In this study, in vivo labeling with a single pulse of tritiated thymidine after partial duct obstruction was used to ascertain whether newly formed islet cells were in fact derived from cells in the ductal epithelium. Supportive evidence for this formulation was also sought using immunocyto-chemistry for islet hormones and in situ hybridization for glucagon and insulin mRNA to probe areas of proliferating duct cells. Endocrine cell differentiation was observed as a migration of cells out from small ducts beginning at about 10 days after obstruction. Duct and islet cell labeling indices (LI; %) in control animals remained at a low level (0.35 ± 0.01 and 0.36 ± 0.03, respectively) throughout the experiment. In contrast, at 2 weeks after partial obstruction, the duct and islet cell LI were 4.2 ± 0.7 and 0.80 ± 0.1 (p < 0.05 vs. control). After 2 weeks, there was a rapid and significant 86% decline in the duct cell LI to a low of 0.6 ± 0.2 at 8 weeks, which was accompanied by a comparable, but reciprocal, 113% increase in the islet cell LI to a high of 1.7 ± 0.8 (p < 0.05). In situ hybridization demonstrated glucagon and insulin mRNA-positive cells within intralobular ducts as early as 6 and 8 days, respectively, after obstruction. Glucagon and insulin peptides appeared in these cells at ∼8 and 10 days, respectively, as cells migrated out from the duct wall. This study provides additional evidence that further supports our concept that pancreatic endocrine cell differentiation in this model reiterates the normal ontogeny of β cell differentiation from cells in the ductular epithelium.

The Structural Integrity of the Islet In Vitro: The Effect of Incubation Temperature

The purpose of this study was to examine the effect of incubation temperature on the structural integrity of the islet during culture. Islets were isolated from the pancreas of the Syrian golden hamster and cultured in a collagen gel for < or =12 days at 24 degrees C or 37 degrees C. At 24 degrees C, cells in the islet periphery died, leading to a complete disintegration of the mantle region in 37.4+/-5.6% of the islets. In comparison, at 37 degrees C, few islets exhibited mantle disintegration (p<0.001). Insulin immunoreactivity was distributed nonhomogeneously in islets at 24 degrees C, and the intensity of the staining, by using a semiquantitative scale (0-3), was +1. Islets cultured at 37 degrees C had a normal homogeneous distribution of insulin immunoreactivity with a score of +3. As the pancreas is a complex gland composed of different cell types, and cell-cell interactions are known to be important in the maintenance of cell survival, additional experiments were repeated to include the coculture of islets with duct epithelial cells. The proportion of islets that developed mantle disintegration was now reduced to 2.5+/-0.3% (p<0.001), comparable to that seen at 37 degrees C. Similar results were obtained for islets cultured in the presence of duct-conditioned medium (DCM). Together with the preservation of the islet mantle, islets cultured in the presence of duct epithelial cells or DCM had a normal homogeneous distribution of insulin immunoreactivity, with a staining intensity of +3. We conclude that incubation temperature has a profound effect on the structural integrity of islets, and that the detrimental effects of low-temperature culture can be mitigated by coculture of islets with secretory products derived from pancreatic ductal cells. These data provide evidence for a trophic relation between pancreatic islets and ductal epithelium.