Three-dimensional imaging of tumor angiogenesis

OBJECTIVE: To three-dimensionally visualize the microvessel environment of tumor angiogenesis by confocal laser scanning microscopy (CLSM). STUDY DESIGN: To reveal underlying mechanisms of tumor angiogenesis, a 7, 12-dimethylbenz(a) anthracene-induced rat cancer model was used. For demonstrating tumor vasculature, fluorescence injection method (FITC-conjugated gelatin solution) was employed. FITC gelatin was injected into the left ventricle of the rat heart. After complete perfusion, the mammary glands were resected, fixed under ice cold conditions and subjected to immunohistochemistry (IHC) for tumor cells. The LSM-410 (Carl Zeiss, Jena, Germany) was employed on thick sections (300-2,000 microns) to elucidate detailed microvessel networks (MVN) and tumor cells. RESULTS: Tumor vasculature on thick sections was clearly detected by CLSM at the maximum focus depth of 2,000 microns. Three-dimensional (3-D), reconstructed images of normal mammary glands showed regular and linear MVN. In DMBA-induced mammary cancer, vascular density of MVN was markedly increased and showed an anastomosing, irregular MVN pattern. Furthermore, focal segmentation and tortuous, branching patterns of microvessels were also seen. CONCLUSION: Application of the fluorescence injection method and IHC using CLSM was very useful for studying the 3-D relationship between tumor angiogenesis and neoplastic epithelial changes. These results suggest that application of this technique is ideal for studying 3-D imaging of tumor angiogenesis.     

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.     

Ontogeny of hormone-secreting cells of the rat pituitary gland: An immunocytochemical study on dissociated cells

Summary An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18–21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80–95% of the cells were immunoreactive.     

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.     

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β–TSH, β–FSH, β–LH, ACTH, and α–MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.     

In Vitro Recapitulation of Functional Microvessels for the Study of Endothelial Shear Response,Nitric Oxide and [Ca2+]i

Microfluidic technologies enable in vitro studies to closely simulate in vivo microvessel environment with complexity. Such method overcomes certain constrains of the statically cultured endothelial monolayers and enables the cells grow under physiological range of shear flow with geometry similar to microvessels in vivo. However, there are still existing knowledge gaps and lack of convincing evidence to demonstrate and quantify key biological features of the microfluidic microvessels. In this paper, using advanced micromanufacturing and microfluidic technologies, we presented an engineered microvessel model that mimicked the dimensions and network structures of in vivo microvessels with a long-term and continuous perfusion capability, as well as high-resolution and real-time imaging capability. Through direct comparisons with studies conducted in intact microvessels, our results demonstrated that the cultured microvessels formed under perfused conditions recapitulated certain key features of the microvessels in vivo. In particular, primary human umbilical vein endothelial cells were successfully cultured the entire inner surfaces of the microchannel network with well-developed junctions indicated by VE-cadherin staining. The morphological and proliferative responses of endothelial cells to shear stresses were quantified under different flow conditions which was simulated with three-dimensional shear dependent numerical flow model. Furthermore, we successfully measured agonist-induced changes in intracellular Ca²⁺ concentration and nitric oxide production at individual endothelial cell levels using fluorescence imaging. The results were comparable to those derived from individually perfused intact venules. With in vivo validation of its functionalities, our microfluidic model demonstrates a great potential for biological applications and bridges the gaps between in vitro and in vivo microvascular research.     

Effect of the growth hormone-secreting tumor StW5 on pituitary and adrenal gland function in rats

A growth hormone-secreting tumor (StW5 was implanted into male rats and resulted in a tripling of adrenal weight concomitant with a 30% decrement in pituitary weight. Plasma concentrations of corticosterone in tumor-bearing (TB) rats were significantly elevated at rest or after ACTH injections or the stress of either anesthesia. The rise in plasma concentrations of corticosterone was due mainly to the large increment in adrenal size although a significant increase in adrenal responsiveness to ACTH was demonstrated in vitro. In addition, plasma corticosterone concentrations were higher in TB rats despite both a doubling of the blood volume and a 50% increase in liver capacity to metabolize corticosterone. Pituitary ACTH content was significantly lower in TB rats, but these pituitary glands could still release near-normal quantities of ACTH as shown both by in vitro incubations and adrenal corticosterone output following ether stress.     

Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization.

Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 micron to 25 micron and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present.     

Community dynamics, trade-offs, invasion criteria and the evolution of host resistance to microparasites

A hybrid cellular automaton model is described and used to simulate early tumor growth and examine the roles of host tissue vascular density and tumor metabolism in the ability of a small number of monoclonal transformed cells to develop into an invasive tumor. The model incorporates normal cells, tumor cells, necrotic or empty space, and a random network of native microvessels as components of a cellular automaton state vector. Diffusion of glucose and H(+)ions (the latter largely resulting from the tumor's excessive reliance on anaerobic metabolism) to and from the microvessels, and their utilization or production by cells, is modeled through the solution of differential equations. In this way, the cells and microvessels affect the extracellular concentrations of glucose and H(+)which, in turn, affect the evolution of the automaton. Simulations of the model demonstrate that: (i) high tumor H(+)ion production is favorable for tumor growth and invasion; however for every H(+)ion production rate, there exists a range of optimal microvessel densities (leading to a local pH favorable to tumor but not to normal cells) for which growth and invasion is most effective, (ii) at vascular densities below this range, both tumor and normal cells die due to excessively low pH, (iii) for vascular densities above the optimal range the microvessel network is highly efficient at removing acid and therefore the tumor cells lose their advantage over normal cells gained by high local H(+)concentration. While significant spatial gradients of glucose formed, no regions of detrimentally poor glucose perfusion (for either cell type) were observed, regardless of microvessel density. Depending on metabolic phenotype, a variety of tumor morphologies similar to those clinically observed were realized in the simulations. Lastly, a sharp transition (analogous to that of the adenoma-carcinoma sequence) between states of initial tumor confinement and efficient invasiveness was observed when H(+)production reached a critical value.     

Patterned vascularization in a directional ice‐templated scaffold of decellularized matrix

Vascularization is fundamental for large‐scale tissue engineering. Most of the current vascularization strategies including microfluidics and three‐dimensional (3D) printing aim to precisely fabricate microchannels for individual microvessels. However, few studies have examined the remodeling capacity of the microvessels in the engineered constructs, which is important for transplantation in vivo. Here we present a method for patterning microvessels in a directional ice‐templated scaffold of decellularized porcine kidney extracellular matrix. The aligned microchannels made by directional ice templating allowed for fast and efficient cell seeding. The pure decellularized matrix without any fixatives or cross‐linkers maximized the potential of tissue remodeling. Dramatical microvascular remodeling happened in the scaffold in 2 weeks, from small primary microvessel segments to long patterned microvessels. The majority of the microvessels were aligned in parallel and interconnected with each other to form a network. This method is compatible with other engineering techniques, such as microfluidics and 3D printing, and multiple cell types can be co‐cultured to make complex vascularized tissue and organ models.     

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.     

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.     

Cerebral microvessels and derived cells in tissue culture: Isolation and preliminary characterization

Summary Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 μm to 25 μm and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present. This work was supported in part by an Arteriosclerosis Specialized center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health (HL-14230) and Grant 584-127703 from the Veterans Administration.     

Aquaporin 1 is important for maintaining secretory granule biogenesis in endocrine cells

Aquaporins (AQPs), a family of water channels expressed in epithelial cells, function to transport water in a bidirectional manner to facilitate transepithelial fluid absorption and secretion. Additionally, AQP1 and AQP5 are found in pancreatic zymogen granules and synaptic vesicles and are involved in vesicle swelling and exocytosis in exocrine cells and neurons. Here, we show AQP1 is in dense-core secretory granule (DCSG) membranes of endocrine tissue: pituitary and adrenal medulla. The need for AQP1 in endocrine cell function was examined by stable transfection of AQP1 antisense RNA into AtT20 cells, a pituitary cell line, to down-regulate AQP1 expression. These AQP1-deficient cells showed more than 60% depletion of DCSGs and significantly decreased DCSG protein levels, including proopiomelanocotin/pro-ATCH and prohormone convertase 1/3, but not non-DCSG proteins. Pulse-chase studies revealed that whereas DCSG protein synthesis was unaffected, approximately 50% of the newly synthesized proopiomelanocortin was degraded within 1 h. Low levels of ACTH were released upon stimulation, indicating that the small number of DCSGs that were made in the presence of the residual AQP1 were functionally competent for exocytosis. Analysis of anterior pituitaries from AQP1 knockout mice showed reduced prohormone convertase 1/3, carboxypeptidase E, and ACTH levels compared to wild-type mice demonstrating that our results observed in AtT20 cells can be extended to the animal model. Thus, AQP1 is important for maintaining DCSG biogenesis and normal levels of hormone secretion in pituitary endocrine cells.     

Pituitary ACTH responsiveness in the vasopressin deficient rat.

ACTH concentration and the responsiveness of dispersed anterior pituitary cells to hypothalamic extract and to vasopressin were studied in homozygous (DI), heterozygous (HTZ), DI-pitressin treated (DIP) Brattleboro rats, and in control Long-Evans rats.Absolute, but not relative, anterior pituitary weights of HTZ, DI, and DIP animals were significantly smaller than those of controls. The concentration of immunoreactive (I) and bioreactive (B) ACTH in dispersed anterior pituitary cells was greater in DI and DIP than in HTZ or in control animals, although the total amount of ACTH was greater in control than DI or HTZ animals. Media from incubates of pituitary cells derived from DI and DIP animals contained less I and B ACTH than those from HTZ or control animals. Pituitary cells derived from DI animals secreted markedly less ACTH following incubation with hypothalamic extract (NIH-HE-RP-1) than did cells from HTZ animals. The response in DIP animals was intermediate between that of DI and HTZ animals. In contrast, pituitary cells derived from DI and DIP animals were significantly more responsive to vasopressin than those from control or HTZ animals.     

Immunohistochemical localization of huntingtin-associated protein 1 in endocrine system of the rat.

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.     

Leukocyte-platelet aggregate adhesion and vascular permeability in intact microvessels: role of activated endothelial cells

Leukocyte-platelet aggregation and aggregate adhesion have been indicated as biomarkers of the severity of tissue injury during inflammation or ischemic reperfusion. The objective of this study is to investigate the mechanisms of the aggregate adhesion and quantitatively evaluate its relationship with microvessel permeability. A combined autologous blood perfusion with single microvessel perfusion technique was employed in rat mesenteric venular microvessels. The aggregate adhesion was induced by systemic application of TNF-alpha plus local application of platelet-activating factor (PAF). Changes in permeability were determined by measurements of hydraulic conductivity (Lp) before and after aggregate adhesion in the same individually perfused microvessels. The compositions of the adherent aggregates were identified with fluorescent labeling and confocal imaging. In contrast to leukocyte adhesion as single cells resulting in no increase in microvessel permeability, aggregate adhesion induced prolonged increases in microvessel Lp (6.1 +/- 0.9 times the control, n = 9) indicated by the initial Lp measurements after 3 h of blood perfusion, which is distinct from the transient Lp increase caused by PAF-induced endothelial activation in the absence of blood. Isoproteronol (Iso) attenuated aggregate adhesion-mediated Lp increases if applied after autologous blood perfusion and prevented the aggregate adhesion if the initial endothelial activation is inhibited by applying Iso before PAF administration but showed less effect on single leukocyte adhesion. This study demonstrated that leukocyte-platelet aggregate adhesion via a mechanism different from that of single leukocyte adhesion caused a prolonged increase in microvessel permeability. Our results also indicate that the initial activation of endothelial cells by PAF plays a crucial role in the initiation of leukocyte-platelet aggregate adhesion.     

Forskolin Stimulates Adenylate Cyclase Activity, Cyclic AMP Accumulation, and Adrenocorticotropin Secretion from Mouse Anterior Pituitary Tumor Cells

Abstract: The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/D16-16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nu-cleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.     

Lead-haematoxylin as a stain for endocrine cells

Summary A modification of MacConaill's lead-haematoxylin has been found to stain several endocrine cells producing polypeptides and monoamines, particularly A and D cells of the pancreatic islet, thyroid C cells, gastro-intestinal enterochromaffin cells, gastric G and X cells, pituitary ACTH and MSH cells, adrenal medullary cells, and chemoreceptive cells of the carotid body. A careful comparison of the results of this method with those of HCI-basic dye method and of monoamine methods suggested that carboxyl groups of proteins may be the main binding site of lead-haematoxylin. Experiments with various pretreatments of tissue sections support such a hypothesis. The possibility that biogenic amines take also some part in the staining cannot be ruled out.