Neoplastic modulation of extracellular matrix: proteoglycan changes in the rabbit mesentery induced by V2 carcinoma cells.

Previous studies have shown that the invasion of V2 carcinoma cells in the rabbit mesentery is associated with marked extracellular matrix synthesis leading eventually to an overall increase in mesenteric mass. The purpose of the present study was to investigate the structural and biochemical composition of the extracellular matrix in tumor-free parts of rabbit mesenteries at various stages after intraperitoneal implantation of V2 carcinoma cells. The overall thickness of the tumor-implanted mesenteries increased progressively and peaked at about Day 14, when it was about 8 times greater than the untreated or liver-implanted controls. This was mainly the result of an accumulation of extracellular matrix components. In particular, there was a marked increase in both collagen fibers and proteoglycan granules, as well as filaments, probably hyaluronic acid, as visualized by ruthenium hexammine trichloride. Stereological analysis showed a 6-fold increase in collagen fibers and a significant increase in the density and average diameter of proteoglycan granules. Biochemical analysis revealed a marked elevation in uronic acid content in the tumor-implanted mesenteries. Specifically, they contained 2.6 and 8.6 times the amount of hyaluronic acid and chondroitin sulfate, respectively, than did controls. Furthermore, the relative percentage of chondroitin sulfate was elevated markedly (26 versus 6% in controls). However, the content of heparan or dermatan sulfate did not vary significantly. Stereological analysis of the fibroblasts showed that their absolute number had doubled and that the cell volume of the individual fibroblast had increased markedly. This suggests that the fibroblasts were responsible for the excessive production of the extracellular matrix. These results support the concept that carcinoma cells can modulate their surrounding extracellular environment by stimulating the synthesis of connective tissue in the host mesenchymal cells.

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography.

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.

A routine method for contrasting elastin at the ultrastructural level.

A reliable, simple, and inexpensive method for ultrastructural investigation of elastin is described. This method uses uranyl acetate dissolved in absolute methanol, followed by an optional lead citrate counterstain. The procedure was tested on a number of animal and human tissues that had been fixed and processed differently.

Mesenterial-window hyperplasia in the lactating rat.

By examining the true mesentery adjacent to the small gut before and after weaning, as well as in age-matched controls, we found that the number of mesenteric windows, their total area, and their total DNA content increased significantly during lactation. Simultaneously, Feulgen-DNA absorption analysis in individual mesenteric cells showed that these had a normal DNA stemline, and a normal distribution within the G1-G2 range. The hyperplasia and growth developed and declined temporally somewhat parallel in the mesenterial windows and their adjacent small gut, suggesting that an as yet unknown common factor(s) governs the hyperplastic growth in both these tissues. The novel physiological, mesenterial-window hyperplasia in the lactating rat described in the present study may prove useful for studies of the mesenteric function and the regulation of cell proliferation.

Hyperplasia of the mesenterial windows precedes that of the small gut in the streptozotocin-diabetic rat.

Hyperplastic growth of the mesenterial windows abutting the small gut occurs in lactating rats (Bergström and Norrby 1988) and chronically diabetic rats (Norrby et al. 1983). In the present study, early events in the mesenterial windows and the small gut in streptozotocin-diabetic rats were examined. The area of the mesenterial windows had already increased significantly on day 1 and hyperplasia in terms of increased DNA content, as well as an increase in histamine content (a mast-cell marker), was established from day 2 of diabetes. The increase in total mesenterial window content of DNA, histamine and protein was roughly linear and parallel from day 2 to day 19. The small-gut circumference increased transiently on day 1, but the small-gut mucosal volume was unaffected on days 1 and 2. The small-gut wet weight increased significantly from day 5, whereas elongation was not observed until day 19. The difference in time between the appearance of hyperplasia and the growth of the mesenterial windows and their adjoining gut and the rate with which the hyperplasia proceeds in the two tissues indicate that the regulatory mechanisms of early hyperplastic growth in these tissues are not identical. The factor(s) causing mesenterial window growth and hyperplasia is/are as yet unknown.

[Comparative characteristics of plasma lipoproteins and mesenteric lymph in the rabbit: spectrum and chemical properties]. / Sravnitel'naia kharakteristika lipoproteidov plazmy krovi i mezenerial'noi limfy krolika: spektr i khimicheskii sostav.

Rabbit mesenteric lymph contained all the main classes of lipoproteins detected in plasma: chylomicrons (CM), very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL). The main classes of lipoproteins in lymph were CM and VLDL, whereas LDL and HDL were the main lipoproteins in blood plasma. The heaviest subfractions HDL3 were not observed in mesenteric lymph. Lymph contained only low amount of LDL; molecular mass of lymphatic LDL was lower as compared with the blood plasma lipoproteins. Mesenteric lymph lipoproteins were enriched with triglycerides and contained less cholesterol, phospholipids and protein than the corresponding blood plasma lipoproteins. As distinct from blood plasma lipoproteins, apo E and apoproteins of low molecular mass (apo C?) were not found in lymphatic lipoproteins. Content of Apo-I was much higher in lymphatic VLDL as compared with the blood plasma VLDL. The dissimilarity in patterns and composition between blood plasma and lymphatic lipoproteins indicated that the latter were derived from intestine. Thus, intestine was able to produce CM, VLDL, HDL2 and apparently a small amount of LDL.

Distribution of polyanionic sites in the developing gonads and the dorsal mesentery of the chick embryo.

The distribution of glycoconjugates was investigated in the embryonic trunk mesoderm used as a substrate by migrating primordial germ cells (PGCs) by means of ultrastructural cytochemistry. In both mesentery and developing gonads polyanionic sites were abundant in epithelial and mesenchymal cell coats, basal laminae, and extracellular matrices (ECM). In the latter, polyanions distributed on microfibrils and granules were associated with collagen fibers, forming an entangled network. No preferential association of this fibrillo-granular material with PGCs was observed, suggesting that polyanions present in ECM likely act by promoting inflation of the extracellular spaces rather than by providing mechanical guides for the moving cells.

Isolation of fatty acid amide as an angiogenic principle from bovine mesentery.

Erucamide (13-docosenamide) was found to be the major bovine mesentery angiogenic lipid as assessed by chorioallantoic membrane (CAM) assay. Two micrograms of this lipid caused angiogenesis in the assay. Angiogenic activity of this naturally occurring lipid was also found by rat corneal micropocket and mouse dorsal air-sac assays. Specificity of the chemical structure which elicited activity was low, however. The mechanism of angiogenic activity is unknown and this lipid does not promote proliferation of endothelial cells or induce inflammatory effects.

A morphologic and histochemical study of the mesentery in the guinea pig.

The guinea pig mesentery is a uniform, continuous, thin (18 micron) sheet of connective tissue covered by a single layer of flattened mesothelial cells on both surfaces. Tight and gap junctions provide for cell-to-cell adhesion among mesothelial cells. These cells possess numerous micropinocytotic vesicles; a conspicuous basal lamina separates the mesothelium from the underlying connective tissue. Most of the material found between the two serous coverings consisted of a three-dimensional meshwork of abundant collagenous fibers intermingled with a sparse net of very thin (0.4 micron) elastic fibers. Two distinct populations of collagen fibrils are segregated into different compartments of the mesentery. One population is formed of thick (56 nm) fibrils which associate to form closely packed fibers. The second population, composed of loosely arranged thin (38 nm) fibrils which do not become assembled into fibers, is found underlying the basal lamina that separates the mesothelium from the connective tissue. These observations strongly suggest that the mesentery contains both collagens type I and type III. The guinea pig mesentery contains 6.8 mg of sulfated glycosaminoglycans/g dry weight. Most of these glycosaminoglycans (78%) were identified as dermatan sulfate, whilst the rest (22%) corresponded to heparan sulfate.

Hyperplasia and growth of the true mesentery in the diabetic rat.

The true mesentery was studied in rats 4 weeks after they had been rendered diabetic with streptozotocin. The diabetic animals showed elongation and enlargement of the small intestine despite reduced body growth compared with controls of the same age. The mesentery in diabetic animals showed increased total area and contained an increased number of "windows", but the increment of total protein, DNA, and histamine (a marker of mast cells) was non-uniform and less than the increase in area. There appeared to be a close relationship between hyperplasia of the small intestine and its mesentery. The number of mast cells yielded by peritoneal lavage was increased in the diabetics. We suggest that our observation of the hyperplastic mesenteric reaction in diabetic rats may make a useful model for the study of growth, profiferation, and function of the mesentery available.

Variations in levels of cyclic AMP and GMP in the thymus, spleen and mesenteric lymph node of rats after the induction of adjuvant arthritis.

The development of adjuvant arthritis induced important modifications in intracellular levels of cyclic AMP and GMP in primary and secondary lymphoid organs: a continuous decrease in cyclic AMP and a biphasic increase in the level of cyclic GMP which correlate well with the onset of the acute phase and the systemization of the disease.

Decrease in peritoneal mast cell count in rats with adjuvant arthritis. I. Time course and interstrain difference.

Adjuvant was injected into the left hind paw of Sprague-Dawley (SD) and Wistar rats. In SD rats a marked decrease in the mast-cell count and histamine content of the peritoneal fluid and a significant increase in the histamine content as well as the mast-cell count in the mesentery appeared between 1 and 2 weeks after adjuvant injection. This period approximately coincided with that of the development of secondary inflammatory lesions such as polyarthritis. The adjuvant-induced changes in the peritoneal fluid were more marked in SD than in Wistar rats, corresponding to more intense adjuvant arthritis in the SD strain. In Wistar rats a tendency towards an increase in the histamine content of the mesentery was observed from 2 weeks after adjuvant injection. Adjuvant injection did not have any significant effect on the histamine content of the caecum or the skin of either strain. These results show that adjuvant causes a redistribution of mast cells between the peritoneal fluid and the tissues surrounding the peritoneal cavity probably as a result of delayed hypersensitivity.

The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts.

The reactions of glyoxylic acid with peripheral stores of noradrenaline and 5-hydroxytryptamine to provide a fluorescence histochemical method for their localization have been investigated. Incubation in glyoxylic acid, followed by drying and heating of whole mount preparations gives an intense and well localized reaction. For incubation, a concentration of 2% glyoxylic acid, buffered to pH 7 at room temperature for 30 minutes gives ideal results. The method is equally good if the pH is varied in the range 6 to 9 or if the tissue is stored in the incubation mixture for up to 6 hours. Ideal development of the fluorophore requires an initial excess of moisture in the tissue, that this moisture is driven off during development, and that the tissue is protected from further moistening. A suitable method of achieving these ends is to heat partially dried tissue at 100 degrees C for 4 minutes and then cover it with paraffin oil. 5-hydroxytryptamine can be readily distinguished from noradrenaline because it forms a fluorophore after reaction at pH 3.5, whereas noradrenaline does not. Both amines can be visualized after incubation at neutral pH. Comparison with the formaldehyde vapour technique reveals three main advantages (and no disadvantages) of the glyoxylic acid method: (1) it gives a finer localization with higher fluorescence yield, (2) the glyoxylic acid method is less susceptible to variations in procedure and, (3) it is both simpler and quicker to apply.

Comparative analysis of protein content in rat mesenteric tissue, peritoneal fluid, and plasma.

Albumin, transferrin, and total protein concentrations were measured in the mesenteric tissue, peritoneal fluid, and plasma of 12 ketamine-Nembutal-anesthetized Sprague-Dawley rats. Tissue samples were obtained with an 8-mm trephine; tissue water content was determined by a microgravimetric method to be 5.2 +/- 0.3 microgram water/microgram dry wt. Peritoneal fluid was collected by capillary action in hematocrit tubes, and blood samples were taken from a femoral artery catheter. Total protein concentrations of plasma (5.8 +/- 0.3 g/dl) and peritoneal fluid (2.6 +/- 0.1 g/dl) were determined by Lowry assay. Ratios of peritoneal fluid and tissue densitogram areas to plasma area were used to calculate total protein content of peritoneal fluid (2.5 +/- 0.1 g/dl) and tissue (1.8 +/- 0.2 g/dl). Albumin concentrations were 1.1 +/- 0.1 g/dl for tissue, 1.4 +/- 0.1 g/dl for peritoneal fluid, and 2.8 +/- 0.1 g/dl for plasma. Transferrin concentrations were 0.09 +/- 0.01 g/dl for tissue, 0.13 +/- 0.01 g/dl for peritoneal fluid, and 0.28 +/- 0.01 g/dl for plasma. Peritoneal fluid protein concentrations were similar to values found for lymph in previous studies. Protein concentration in the tissue buttons was significantly less than that of peritoneal fluid. This contradicts the widely held assumption that the protein concentration of fluid outside the matrix is representative of a well-mixed interstitial matrix fluid protein concentration.

The radial protein concentration profile in the interstitial space of the rat ileal mesentery.

The radial distribution of the protein concentration in the interstitium between arteriolar and venular vessels of the ileal mesentery of the rat was examined. Protein mass was determined by means of uv ultramicrospectrophotometry (UMS) and the relative volume distribution by means of fluorescence microscopy (FM) using the Na fluorescein and FITC-dextran (10,000 mol wt). UMS revealed gradients for protein mass from the vessels out into the interstitial space. FM showed a uniform distribution of fluorescence in the interstitium between the vessels. A gradient for protein mass without a gradient for volume distribution signifies the presence of a concentration gradient for protein in the interstitial space. The protein concentration across the arteriolar wall drops from 5.4 +/- 0.24 (SD) to 2.6 +/- 0.65% and across the venular wall from 5.4 +/- 0.24 to 3.3 +/- 0.43%. From the perivascular site the protein concentration declines exponentially reaching a minimum average interstitial concentration of 1.6 +/- 0.56%. Minimal protein concentration occurred at a point 37 +/- 6.4% of the 295 +/- 37 micron distance from the arteriolar to the venular vessels. In view of this distribution, it is unlikely that lymph or direct samples of interstitial fluid are representative of the perivascular protein concentration.

The effect of tranylcypromine on levels of 6-keto-PGF1 alpha and other prostaglandins in brain and mesentery of the mouse.

Tranylcypromine (TCP), which can inhibit prostacyclin (PGI2) synthesis in vitro, has been shown to facilitate platelet aggregation in damaged cerebral arterioles of the mouse when given intraperitoneally (50 mg/kg) one hour before inducing aggregation. The same dose has no effect on platelet aggregation in damaged mesenteric arterioles. The present experiments used HPLC and GC/MS to analyze PG levels and show that 5 or 50 mg/kg TCP, given intraperitoneally one hour before sacrificing the mouse, moderately reduces the level of 6-keto-PGF1 alpha, the stable metabolite of PGI2, in incubated brain homogenates. This finding supports the hypothesis that TCP's enhancement of platelet aggregation in the brain was affected by a reduction in PGI2 levels. When 500 micrograms/ml TCP was added to the incubate of brain homogenate from mice given 50 mg/kg, PGE2 levels were reduced as well as the levels of 6-keto-PGF1 alpha. In incubated mesentery, the level of 6-keto-PGF1 alpha was also reduced by treating mice with 50 mg/kg TCP. The latter result failed to support the hypothesis that levels of mesenteric 6-keto-PGF1 alpha would be unaltered by TCP in parallel with the inability of TCP to alter platelet aggregation in mesenteric arterioles. Thus our data fails to support an overall hypothesis relating TCP action on platelet aggregation to its inhibitory effect on PGI2 synthesis. At the same time the data do not rule out such a relationship for mouse brain.