Isolation and characterisation of CD9-positive pituitary adult stem/progenitor cells in rats.

S100ß protein and SOX2-double positive (S100ß/SOX2-positive) cells have been suggested to be adult pituitary stem/progenitor cells exhibiting plasticity and multipotency. The aim of the present study was to isolate S100ß/SOX2-positive cells from the adult anterior lobes of rats using a specific antibody against a novel membrane marker and to study their characteristics in vitro. We found that cluster of differentiation (CD) 9 is expressed in the majority of adult rat S100ß/SOX2-positive cells, and we succeeded in isolating CD9-positive cells using an anti-CD9 antibody with a pluriBead-cascade cell isolation system. Cultivation of these cells showed their capacity to differentiate into endothelial cells via bone morphogenetic protein signalling. By using the anterior lobes of prolactinoma model rats, the localisation of CD9-positive cells was confirmed in the tumour-induced neovascularisation region. Thus, the present study provides novel insights into adult pituitary stem/progenitor cells involved in the vascularisation of the anterior lobe.

Microvessel density and VEGF expression in pituitaries of pregnant women.

OBJECTIVE: In pregnant women, the pituitary is enlarged and the prolactin (PRL) secreting cells increase in size and number. This PRL cell hyperplasia is associated with hyperprolactinemia. The aim of the present work was to investigate adenohypophysial vascularization and immunoexpression of vascular endothelial growth factor (VEGF) in pituitaries of pregnant and post-partum women and compare the results with age-matched adenohypophyses of nonpregnant women who had no endocrine diseases. DESIGN: Pituitaries (n=18) obtained by autopsy from female patients of reproductive age who had died during pregnancy, after abortion or during post-partum were immunostained for CD-34 and VEGF using the streptavidinbiotin- peroxidase complex method. RESULTS: The results showed that microvessel densities and VEGF immunoexpression in the adenohypophyses of pregnant and post-partum women were similar to those found in the control pituitaries. CONCLUSION: It can be concluded that pituitary enlargement and PRL cell hyperplasia in pregnant women may occur without neovascularization and increased VEGF immunoexpression.

Spindle cell oncocytoma of the adenohypophysis with marked hypervascularity. Case report.

A 68-year-old male presented with a very rare case of spindle cell oncocytoma (SCO), a recently identified very rare neoplasm of the anterior pituitary, manifesting as panhypopituitarism and visual field defect. The pituitary tumor with suprasellar extension was only partially resected via transsphenoidal surgery because of the tumor consistency and bleeding. Histological diagnosis was consistent with schwannoma. The tumor regrew and angiography revealed hypervascularity, so a transcranial approach was employed for the re-operation which only achieved partial resection because of intraoperative extensive bleeding. The tumor cells showed similar histological and immunohistochemical profiles to the previous specimen, but electron microscopy demonstrated that cytoplasm abundantly filled with mitochondria. The final diagnosis of SCO was established and the patient received postoperative conventional radiation therapy of 50 Gy. Only 15 cases of SCO have been reported, and the diagnosis was mistaken in many cases as schwannoma, oncocytic pituitary adenoma, or craniopharyngioma, and multiple surgeries followed by radiation therapy were required.

Anterior pituitary cell networks.

Both endocrine and non-endocrine cells of the pituitary gland are organized into structural and functional networks which are formed during embryonic development but which may be modified throughout life. Structural mapping of the various endocrine cell types has highlighted the existence of distinct network motifs and relationships with the vasculature which may relate to temporal differences in their output. Functional characterization of the network activity of growth hormone and prolactin cells has revealed a role for cell organization in gene regulation, the plasticity of pituitary hormone output and remarkably the ability to memorize altered demand. As such, the description of these endocrine cell networks alters the concept of the pituitary from a gland which simply responds to external regulation to that of an oscillator which may memorize information and constantly adapt its coordinated networks' responses to the flow of hypothalamic inputs.

Transmission and scanning electron microscopy study of the characteristics and morphology of pericytes and novel desmin-immunopositive perivascular cells before and after castration in rat anterior pituitary gland.

Pericytes are perivascular cells associated with microcirculation. Typically, they are localized close to the capillary wall, underneath the basement membrane, and have sparse cytoplasm and poorly developed cell organelles. However, the specific properties of pericytes vary by organ and the conditions within organs. We recently demonstrated that pericytes in rat anterior pituitary gland produce type I and III collagens. The present study attempted to determine the morphological characteristics of these pituitary pericytes. Castrated rats were used as a model of hormonal and vascular changes in the gland. Pericytes, as determined by desmin immunohistochemistry, were more numerous and stained more intensely in castrated rats. Transmission electron microscopy revealed that pituitary pericytes displayed the typical characteristics of pericytes. In pituitary sections from castrated rats, the Golgi apparatus of pericytes was well developed and the rough endoplasmic reticulum was elongated. Additionally, scanning electron microscopy revealed four pericyte shapes: oval, elongate, triangular, and multiangular. As compared with normal rats, the proportion of oval pericytes was lower, and the proportions of the other three shapes were higher, in castrated rats. These results suggest that pericytes change their fine structure and cell shape in response to hormonal and vascular changes in the anterior pituitary gland. In addition, a novel type of perivascular cell was found by desmin immunoelectron microscopy. The morphological properties of these cells were dissimilar to those of pericytes. The cells were localized in the perivascular space, had no basement membrane, and contained dilated rough endoplasmic reticulum. This new cell type will require further study of its origin and characteristics.

Functional importance of blood flow dynamics and partial oxygen pressure in the anterior pituitary.

The pulsatile release of hormone is obligatory for the control of a range of important body homeostatic functions. To generate these pulses, endocrine organs have developed finely regulated mechanisms to modulate blood flow both to meet the metabolic demand associated with intense endocrine cell activity and to ensure the temporally precise uptake of secreted hormone into the bloodstream. With a particular focus on the pituitary gland as a model system, we review here the importance of the interplay between blood flow regulation and oxygen tensions in the functioning of endocrine systems, and the known regulatory signals involved in the modification of flow patterns under both normal physiological and pathological conditions.

Effect of estrogen on the blood supply of pituitary autografts in rats.

Estrogens are known to cause pituitary enlargement and lactotroph proliferation. They also modulate pituitary angiogenesis and induce tumor formation. Pituitary grafts, due to the loss of hypothalamic dopamine, also show lactotroph hyperplasia. We investigated the role of estrogen on rat pituitary autograft vascularization by light and transmission electron microscopy, and assessed prolactin (PRL) blood levels, microvessel density (MVD) and cell proliferation using the BrdU labeling index. All adenohypophysial cell types were identified by immunohistochemistry (streptavidin-biotin-peroxidase complex method). The proangiogenic factors, vascular endothelial growth factor (VEGF), its receptor Flk-1, and hypoxia inducible factor-1alpha (HIF-1alpha) were similarly demonstrated. The prevalence of lactotrophs, as well as more intense staining for VEGF, Flk-1 and HIF-1alpha, was noted in those grafts exposed to estrogen, mainly in the area surrounding the central necrotic core. Immunostaining showed Flk-1 expression increased in endothelial cells of the estrogen-exposed grafts as compared with those unexposed. In contrast to the grafts not exposed to estrogen, in the estrogen-exposed grafts, only fenestrated endothelium could be demonstrated, suggesting that estrogen induces fenestration of newly formed capillaries. There was an increase in blood PRL levels in the estrogen-treated groups as compared with controls. Both MVD and BrdU labeling indices were higher in grafts exposed to estrogen, especially after 4 weeks. Our results suggest that estrogen administration not only enhances the expression of proangiogenic factors in the pituitary grafts but also induces their expression at earlier stages, leading to rapid neoformation of purely fenestrated capillaries.

Immunohistochemical expression of nestin in adenohypophysial vessels during development of pituitary infarction.

OBJECTIVES: The aim of this work was to investigate the immunohistochemical expression of nestin, a member of the intermediate filament family, in adenohypophysial vasculature during development and progression of pituitary infarction. METHODS: Forty-five nontumorous adenohypophyses and 34 pituitary adenomas of various types, all exhibiting acute or healing infarcts, were examined immunohistochemically using the streptavidin-biotin-peroxidase complex method. RESULTS: In both adenohypophyses and pituitary adenomas without infarction, nestin was expressed in only a few capillaries and endothelial cells. In acute infarcts without a vascular response, no nestin was demonstrable within necrotic capillaries (50 cases). In organizing infarcts, newly formed vessels spreading into necrotic zones showed nestin expression in all capillaries and practically every endothelial cell (25 cases). In the hypocellular, fibrotic scar phase, only a few vessels (4) were apparent, and immunoreactivity was focal and mild. CONCLUSIONS: Nestin is strongly expressed in newly formed capillaries and is downregulated when infarcts transform to fibrous tissue. Nestin expression may provide valuable insight into the process of pituitary angiogenesis.

Histologic study of the human pituitary gland in acute traumatic brain injury.

PURPOSE: Approximately 25% of patients with traumatic brain injury (TBI) may develop partial or complete hypopituitarism. The causative mechanisms involved in its development are not clear. To the authors' knowledge, there have been no recent morphologic studies of the pituitary following TBI. METHODS: To characterize the resultant histologic changes, this study investigated the pituitaries of 42 patients who died following a motor vehicle accident, all from the Mayo Tissue Registry. Twelve patients died instantly at the scene of the accident (Group I) whereas 30 survived between 3 hours and 7 days (Group II). All pituitary specimens were obtained at autopsy, formalin-fixed and paraffin-embedded. Hematoxylin-eosin sections cut in horizontal or sagittal plane were examined light-microscopically. RESULTS: No infarction was noted in the pituitary specimens from group I. In group II, 13 of 30 (43%) showed acute infarcts of varying size. The extent of infarction in group II ranged from focal to sub-total necrosis involving 90% of the adenohypophysis. CONCLUSIONS: Underlying adenohypophysial pathology in patients dying after TBI is acute infarction. Loss of large numbers of adenohypophysial cells causes reduced secretion of adenohypophysial hormones and may contribute to post-traumatic hypopituitarism.

Inhibition of estrogen-induced pituitary tumor growth and angiogenesis in Fischer 344 rats by the matrix metalloproteinase inhibitor batimastat.

The development of estrogen-induced pituitary prolactinoma in Fischer 344 (F344) rats is associated with enhanced neovascularization. Based on the significance of matrix metalloproteinases (MMPs) for tumor growth and angiogenesis, we have studied the effect of batimastat (BB-94), a synthetic MMPs inhibitor (MMPI) on the progression of prolactin-secreting pituitary adenoma in rats. Pituitary tumors were induced in male F344 rats by s.c. implantation of Silastic tubes containing diethylstilbestrol (DES). The effects of chronic treatment with BB-94 (30 mg/kg b.w.) on pituitary weight, cell proliferation, apoptosis and vascular density were evaluated. We have stated that chronic treatment with batimastat caused a significant reduction in the pituitary weight. Batimastat has been found to decrease cell proliferation evaluated by a number of PCNA-positive stained cell nuclei. A marked increase in the apoptotic index within the pituitary was observed in the study group. Moreover, the density of microvessels identified by CD31 was reduced in the group treated with BB-94. The results of our study provide evidence for an inhibitory effect of batimastat, a synthetic MMPI, on the growth and angiogenesis in an experimental model of human prolactinoma. The ability of BB-94 to suppress established pituitary tumor growth suggests a possible application of MMPIs in the treatment of pituitary adenomas.

Vascularization of rat pituitary autografts.

Pituitary autotransplantation eliminates direct vascular contact between the hypothalamus and the adenohypophysis, and enables us to study the role of the hypothalamus in regulating adenohypophysial endocrine activity. The aim of this study was to investigate vascularization of the pituitary autografts. Three-month-old male Wistar rats were hypophysectomized, and their adenohypophyses were autotransplanted under the renal capsule. The animals were killed 3 weeks after autotransplantation. The grafts were removed and studied by using histology, immunohistochemistry and transmission electron microscopy. In the central portion of the grafts, organizing necrosis was apparent. The peripheral portion of the graft contained all adenohypophysial cell types, with a predominance of lactotrophs. Vascular endothelial growth factor and hypoxia-inducible factor were expressed in the graft mainly in the perinecrotic areas. Several capillaries inside the grafts were lined by continuous unfenestrated epithelium, while others were lined by fenestrated endothelium, suggesting that neovascularization is the result of two processes: ingrowths of capillaries from the renal capsule to the graft, and neoformation of capillaries from pre-existing adenohypophysial vessels. In conclusion, hypoxia seems to be an important factor in the vascularization of pituitary autografts. Mediated via hypoxia-inducible factor, hypoxia stimulates vascular endothelial growth factor secretion, which plays a crucial role in angiogenesis.

Combined CNS and pituitary involvement as a primary manifestation of Wegener granulomatosis.

Wegener granulomatosis (WG) is a systemic vasculitis of small and medium vessels. It predominantly affects the upper and/or lower respiratory airway and kidneys. Its pathogenesis is not fully understood. WG relatively frequently affects the nervous system (in 30-50% according to the different studies). Most frequently, it manifests as necrotizing vasculitis that leads to the peripheral neuropathies or to the cranial nerves palsy. Impairment of the central nervous system (CNS) is less frequent and occurs in 2-8% of patients. Three major pathogenetic mechanisms were described: CNS vasculitis, spreading of granulomas from the adjacent anatomical areas (paranasal cavities, orbit etc.), and new formation of granulomas in brain tissue. This case report describes patients in whom WG manifested in the form of localized skin involvement and combined CNS involvement that included pituitary gland. Atypical presentation of WG impedes and slows down the process of diagnosis and emphasizes the need for collaboration between medical specialists.

Anterior hypopituitarism following traumatic brain injury.

PRIMARY OBJECTIVES: To review evidence that there exists a substantial sub-population of patients with endocrine disorders as a result of traumatic brain injury (TBI) and to underscore the importance of screening patients with TBI considered most at risk for hypopituitarism with the goal of attaining beneficial effects in terms of morbidity and quality of life. DESIGN AND METHODS: Reviewed recent literature regarding the frequency of TBI-induced hypopituitarism. MAIN OUTCOMES AND RESULTS: Studies by Kelly DF, Gaw Gonzalo IT, Cohan P, et al. Hypopituitarism following traumatic brain injury and aneurysmal subarachnoid hemorrhage: A preliminary report. Journal of Neurosurgery 2000;93:743-751, Lieberman SA, Oberoi AL, Gilkison CR, et al. Prevalence of neuroendocrine dysfunction in patients recovering from traumatic brain injury. Journal of Clinical Endocrinology and Metabolism 2001;86:2752-2756 and Aimaretti G, Ambrosio MR, Di Somma C, et al. Traumatic brain injury and subarachnoid haemorrhage are conditions at high risk for hypopituitarism. Screening study at 3 months after the brain injury, In press., found that about one-half to one-third of patients with TBI had anterior pituitary hormone deficiencies, including growth hormone (GH) deficiency in 15-21%, and subtle deficiencies in thyroid, adrenal and gonadal axes. One or more hormonal deficiencies produce diverse physical and psychological symptoms that may mimic symptoms attributed to brain trauma and may impair rehabilitation. A more general concern is the fact that hypopituitarism increases the risk of significant morbidity (e.g. ischaemic heart disease) and mortality (shortened life span). CONCLUSIONS: To attain maximal improvement in mental and physical functioning as well as in quality of life for victims of TBI, it is crucial that anterior pituitary hormonal function be assessed. Appropriate hormone replacement therapy for those patients with both TBI and TBI-induced pituitary function impairment could, for the first time, allow treatment and correction of underlying causes of TBI sequelae rather than merely symptomatic treatment.

Abnormal perfusion of the pituitary gland secondary to dural arteriovenous fistulas in the cavernous sinus: dynamic MR findings.

BACKGROUND AND PURPOSE: If venous congestion is the primary cause of pituitary gland enlargement in cases of dural arteriovenous fistulas (AVFs), other abnormal pituitary findings may be detectable on MR images. We sought to investigate the perfusion abnormality of the pituitary gland secondary to dural AVFs in the cavernous sinus and to clarify its clinical importance. METHODS: Nine consecutive patients (all female; age range, 50-77 years) with dural AVFs in the cavernous sinus underwent prospective MR examinations, including dynamic studies, before and after therapy. Their clinical signs and symptoms were recorded. Two radiologists visually evaluated the enhancement patterns of the anterior pituitary gland. Dynamic MR curves were obtained by locating regions of interest at the center and bilateral peripheral areas of the anterior pituitary gland on coronal images. MR images obtained in five healthy individuals served as controls. RESULTS: No patient had symptoms of hypopituitarism or other endocrine abnormalities. Asymmetric pituitary enhancement was found in five patients; the side with the dural AVF was less enhancing. This finding disappeared after therapy. Although asymmetric enhancement was not detected in the remaining four patients, statistical analysis showed significantly delayed enhancement of the pituitary gland in the patients compared with enhancement patterns in control subjects. After treatment, this delay improved significantly. The pituitary gland significantly decreased in size after treatment. CONCLUSION: Perfusion of the pituitary gland is impaired in patients with a dural AVF in the cavernous sinus. This finding is probably due to venous congestion of the pituitary gland caused by high pressure in the cavernous sinus; it is usually not related to pituitary dysfunction.

Microvascular density and vascular endothelial growth factor expression in normal pituitary tissue and pituitary adenomas.

Microvessel density (MVD) represents a measure of angiogenesis and may be used as an indicator of neoplastic aggressiveness. Vascular endothelial growth factor (VEGF) plays a pivotal role as angiogenic promoter by stimulating endothelial cell proliferation and migration and enhancing vascular permeability. The aim of this study was to investigate MVD and VEGF expression in human pituitary adenomas and normal pituitary gland tissues by immunohistochemistry, and to correlate data with clinical characteristics. Fragments from 46 pituitary adenomas (18 non-functioning, 12 ACTH-secreting, 12 GH-secreting, 4 PRL-secreting) and 19 specimens of normal anterior pituitary gland obtained at surgery were evaluated. MVD in normal anterior pituitary was significantly higher than in tumors (69.2 +/- 28.5 vs 29.3 +/- 19.7; p < 0.0001). Within adenomas, no difference was found in MVD when different histotype, size, sex, age, rate of recurrence or medical pre-surgical treatment were considered. The degree of vascularity was somewhat related only to clinical invasiveness, as evaluated by pre-surgical MRI grading (grade 0 p < 0.05 vs grade 1 and vs grade 2). No statistically significant difference in VEGF expression was found between normal tissue and adenomas and among tumors of different histotype (p = 0.3978). Size, sex, age, rate of recurrence and medical pre-surgical treatment did not influence VEGF expression. No correlation was found between MVD and VEGF expression. In conclusion, MVD was reduced in pituitary adenomas with respect to normal gland. VEGF expression is however well preserved in adenomas and this might contribute to adequate tumoral vascular supply with complex mechanisms other than endothelial cells proliferation.

Increase of gonadotropin-releasing hormone concentration in pituitary portal blood after substance P administration in male rats.

Substance P (SP) affects gonadotropin release from the anterior pituitary gland. In the present study we tested whether SP exerts this effect through GnRH release into pituitary portal blood in intact male rats (INT), orchidectomized rats with s.c. chronically implanted empty Silastic capsule (ORCX), testosterone capsule (ORCX + T), and 17beta-estradiol capsule (ORCX + E2). The pituitary glands were exposed by the transpharyngeal approach under urethane-chloralose anesthesia. Then, the stalk portal vessels were cut and three 30-min portal blood samples were collected. Each first sample of blood was treated as a control before 0.2 ml injection of normal saline, 5 microg, or 25 microg of SP in 0.2 ml of normal saline into the internal carotid artery. GnRH concentration in the purified portal plasma were measured by RIA. Injection of SP into the internal carotid artery caused a significant increase in GnRH concentration in pituitary portal plasma only in INT rats. The higher dose of SP markedly increased GnRH concentration in the 1st blood sample (p < 0.001) and in the 2nd blood sample GnRH concentration was lower but still significant higher than prior SP injection (p < 0.05). The lower dose of SP increased GnRH concentration later, only in the 2nd portal blood sample after intracarotid SP injection (p < 0.001). Injection of normal saline had no effect on GnRH concentration in pituitary portal blood in INT rats. In ORCX, ORCX testosterone- and estrogen-implanted rats portal plasma GnRH concentrations were not changed significantly after injection of both doses of SP. These results indicate that SP stimulates GnRH release into pituitary portal blood and the influence of SP on GnRH neurons depends on the levels of circulating gonadal steroid hormones.

The effect of somatostatin analog octreotide on diethylstilbestrol-induced prolactin secretion, cell proliferation and vascular changes in the rat anterior pituitary gland.

The effects of diethylstilbestrol (DES) and of long-acting somatostatin analog, octreotide (SMS) on the rat anterior pituitary microvasculature have been studied by means of computer-assisted image analysis. Additionally, the effects of DES and SMS on prolactin secretion and anterior pituitary cell proliferation have been studied, as well. The vascularization was visualized using Selye's method modified by Poely et al. (1964). The prolactin serum levels were estimated by radio-immunoassay. The proliferation indices were assessed using bromodeoxyuridine incorporation assay. As expected, it was found that DES sharply increased serum prolactin levels and enhanced cell proliferation in the anterior pituitary gland. DES also induced changes in parameters of vascularization. Simultaneous treatment of rats with SMS inhibited the DES-induced elevation of prolactin levels and pituitary cell proliferation. It also suppressed some but not all DES-induced changes in the anterior pituitary vascularization. These data suggest that the angio-inhibitory activity of SMS might be involved in its anti-tumor action on pituitary adenomas, but not as a sole or principal mechanism.

Over expression of vascular endothelial growth factor and its receptor during the development of estrogen-induced rat pituitary tumors may mediate estrogen-initiated tumor angiogenesis.

Estrogens, which have been associated with several types of human and animal cancers, can induce tumor angiogenesis in the pituitary of Fischer 344 rats. The mechanistic details of tumor angiogenesis induction, during estrogen carcinogenesis, are still unknown. To elucidate the role of estrogen in the regulation of tumor angiogenesis in the pituitary of female rats, the density of blood vessels was analysed using factor VIII related antigen (FVIIIRAg) immunohistochemistry and the expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) was examined by Western blot and immunohistochemical analysis. The expression of VEGF receptor (VEGFR-2/Flk-1/KDR) was also examined by immunohistochemistry. The results demonstrated that 17beta-estradiol (E2) induces neovascularization, as well as the growth and enlargement of blood vessels after 7 days of exposure. The high tumor angiogenic potential was associated with an elevated VEGF/VPF protein expression in the E2 exposed pituitary of ovariectomized (OVEX) rats. VEGF/VPF and FVIIIRAg immunohistochemistry and endothelial specific lectin (UEA1) binding studies, indicate that the elevation of VEGF protein expression initially occurred in both blood vessels and non-endothelial cells. After 15 days of E2 exposure, VEGF/VPF protein expression, in the non-endothelial cell population, sharply declined and was restricted to the blood vessels. The function of non-endothelial-derived VEGF is not clear. Furthermore, immunohistochemical studies demonstrated that VEGFR-2 (flk-1/KDR), expression was elevated significantly in the endothelial cells of microblood vessels after 7 days of E2 exposure. These findings suggest that over expression of VEGF and its receptor (VEGFR-2) may play an important role in the initial step of the regulation of estrogen induced tumor angiogenesis in the rat pituitary.