Seasonal reorganization of hypothalamic neurogenic niche in adult sheep.

Neurogenesis is the process by which new neurons are generated. This process, well established during development, persists in adulthood owing to the presence of neural stem cells (NSCs) localized in specific brain areas called neurogenic niches. Adult neurogenesis has recently been shown to occur in the hypothalamus, a structure involved in the neuroendocrine regulation of reproduction and metabolism, among others. In the adult sheep-a long-lived mammalian model-we have previously reported the existence of such a neurogenic niche located in the hypothalamic arcuate nucleus and the median eminence. In addition, in this seasonal species, the proliferation as well as neuroblasts production varies depending on the time of the year. In the present study, we provide a better characterization of the hypothalamic neurogenic niche by identifying the main components (NSCs, migrating cells, glial cells and blood vessels) using immunohistochemistry for validated markers. Then, we demonstrate the strong sensitivity of these various neurogenic niche components to the season, particularly in the arcuate nucleus. Further, using an electron microscopic approach, we reveal the cellular and cytoarchitectural reorganization of the arcuate nucleus niche following exposure to contrasting seasons. This study provides evidence that the arcuate nucleus and the median eminence contain two independent niches that react differently to the season. In addition, our results support the view that the cytoarchitectural organization of the sheep arcuate nucleus share comparable features with the structure of the subventricular zone in humans and non-human primates.

Miconazole protects blood vessels from MMP9-dependent rupture and hemorrhage.

Hemorrhagic stroke accounts for 10-15% of all strokes and is strongly associated with mortality and morbidity worldwide, but its prevention and therapeutic interventions remain a major challenge. Here, we report the identification of miconazole as a hemorrhagic suppressor by a small-molecule screen in zebrafish. We found that a hypomorphic mutant fn40a, one of several known ß-pix mutant alleles in zebrafish, had the major symptoms of brain hemorrhage, vessel rupture and inflammation as those in hemorrhagic stroke patients. A small-molecule screen with mutant embryos identified the anti-fungal drug miconazole as a potent hemorrhagic suppressor. Miconazole inhibited both brain hemorrhages in zebrafish and mesenteric hemorrhages in rats by decreasing matrix metalloproteinase 9 (MMP9)-dependent vessel rupture. Mechanistically, miconazole downregulated the levels of pErk and Mmp9 to protect vascular integrity in fn40a mutants. Therefore, our findings demonstrate that miconazole protects blood vessels from hemorrhages by downregulating the pERK-MMP9 axis from zebrafish to mammals and shed light on the potential of phenotype-based screens in zebrafish for the discovery of new drug candidates and chemical probes for hemorrhagic stroke.

Ultrastructure of the Subcutaneous Primo-Vascular System in Rat Abdomen.

Recently, we identified the primo-vascular system (PVS), a novel vascular network, in rat subcutaneous tissues. Little is known about the subcutaneous PVS (sc-PVS). Here, we examined the ultrastructure of the sc-PVS in the hypodermis at the rat abdominal midline by electron microscopy. On the surface of sc-PVS, we observed three types of cells: microcells (5-6 µm), large elliptical cells (>20 µm), and erythrocyte (3-4 µm). The inside of the sc-PVS was filled with numerous cells, which can be classified into three major groups: leucocytes, mast cells, and erythrocytes. The dense leucocytes and mast cells were easily noticed. The extracellular matrix of the sc-PVS was mainly composed of extensive fibers (79 ± 6.5 nm) tightly covered by micro- (0.5-1 µm) and nanoparticles (10-100 nm). In conclusion, the ultrastructural features, such as the resident cells on and in the sc-PVS and fiber meshwork covered by particles, indicate that sc-PVS might act as a circulatory channel for the flow and delivery of numerous cells and particles. Our findings will help understand the nature of various sc-PVS beneath-the-skin layers and how they relate to acupuncture meridians.

The blood-tendon barrier: identification and characterisation of a novel tissue barrier in tendon blood vessels.

In the last decade, nanobiotechnology research has emerged as a revolutionising new approach to the 21st century pharmaceutical challenges, offering valuable gains in a vast set of biomedical applications. In the field of bone tissue engineering, a broad range of nanotechnology-based delivery systems have been researched and the most recent developments in high-throughput technology and in silico approaches are creating very high expectations. This review presents a comprehensive overview of the emergent nanotechnology-based materials, processing techniques and research strategies for the delivery of pharmaceutics to bone including the materials general characteristics and the available drug delivery systems to distribute agents systemically or locally. Complementary to what was stated above, it also reviews the latest high-throughput processing techniques and the existent in silico tools (mathematical and computational models) used to help on the design of delivery systems.

Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development.

During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.

Single-dose local simvastatin injection improves implant fixation via increased angiogenesis and bone formation in an ovariectomized rat model.

BACKGROUND: Statins have been reported to promote bone formation. However, taken orally, their bioavailability is low to the bones. Implant therapies require a local repair response, topical application of osteoinductive agents, or biomaterials that promote implant fixation. MATERIAL/METHODS: The present study evaluated the effect of a single local injection of simvastatin on screw fixation in an ovariectomized rat model of osteoporosis. RESULTS: Dual-energy X-ray absorptiometry, micro-computed tomography, histology, and biomechanical tests revealed that 5 and 10 mg simvastatin significantly improved bone mineral density by 18.2% and 22.4%, respectively (P<0.05); increased bone volume fraction by 51.0% and 57.9%, trabecular thickness by 16.4% and 18.9%, trabeculae number by 112.0% and 107.1%, and percentage of osseointegration by 115.7% and 126.3%; and decreased trabeculae separation by 34.1% and 36.6%, respectively (all P<0.01). Bone mineral apposition rate was significantly increased (P<0.01). Furthermore, implant fixation was significantly increased (P<0.05), and bone morphogenetic protein 2 (BMP2) expression was markedly increased. Local injection of a single dose of simvastatin also promoted angiogenesis. Vessel number, volume, thickness, surface area, and vascular volume per tissue volume were significantly increased (all P<0.01). Vascular endothelial growth factor (VEGF), VEGF receptor-2, von Willebrand factor, and platelet endothelial cell adhesion molecule-1 expression were enhanced. CONCLUSIONS: A single local injection of simvastatin significantly increased bone formation, promoted osseointegration, and enhanced implant fixation in ovariectomized rats. The underlying mechanism appears to involve enhanced BMP2 expression and angiogenesis in the target bone.

Comparison of the characteristic features of Bonghan ducts, blood and lymphatic capillaries.

OBJECTIVE: To show that the characteristic morphological and ultrastructural features of a Bonghan corpuscle and duct presented here are consistent with the description given in the early reports of Bonghan Kim. MATERIALS AND METHODS: We compared the morphological aspects of Bonghan ducts with those of blood and lymphatic capillaries on the ultrastructural level to display the manifestly distinctive nature of the Bonghan system. RESULTS: The walls of the ductules were observed to be composed of a single layer of endothelial cells with characteristic rod-shaped nuclei and were not surrounded by a basal lamina or by accessory cells, such as pericytes or smooth muscle cells. The abluminal cell membranes of Bonghan ductules were not attached by anchoring filaments to the fibers of extracellular matrices as observed in lymphatic capillaries. The cytoplasmic processes of ductule endothelial cells appear to form overlapping and interdigitated interconnections which completely lack junctional elements. Although the cytoplasm of ductule endothelial cells contained a well-developed rough endoplasmic reticulum and many free ribosomes and polysomes, there was a relatively small number of pinocytotic vesicles and lacks specific organelles, such as Weibel-Palade bodies. CONCLUSIONS: The Bonghan corpuscles are specialized structures consisting of different types of immune cells randomly scattered as single cells in the matrix or clustered in follicle-like formations. Moreover, the Bonghan ductules in the corpuscle contain flowing immune cells and occasionally basophilic bodies.

Hypothalamic hamartomas associated with epilepsy: ultrastructural features.

Hypothalamic hamartomas (HHs) are associated with a catastrophic form of childhood epilepsy and are intrinsically epileptogenic for the gelastic seizures that are peculiar to this disorder. The cellular mechanisms of seizure generation within HH tissue are unknown. Hypothalamic hamartoma tissue consists of well-differentiated neurons interspersed with glial cells. Based on intrinsic electrophysiologic properties, we hypothesized that small and large HH neurons would have different ultrastructural features. Surgically resected HH tissue samples from 7 patients with refractory epilepsy were studied using electron microscopy. Models of neurons were made from reconstruction of serial sections. Nissl body density, polyribosome density, glycogen density, and the ratio of nuclear area to cytoplasmic area differed significantly between small and large neurons. Abundant small neurons were found in clusters and elaborated terminals that made symmetric, putatively inhibitory, synapses. Symmetric synapses were found predominantly on the soma and proximal dendrites of large projection-like neurons, whereas asymmetric, putatively excitatory, synapses were found primarily on distal dendrites. All samples showed unusual dendritic varicosities. The presence of these morphologically and functionally distinct neurons, the large number of nerve fibers in the neuropil, and the presence of inhibitory and excitatory synapses suggest that HH tissue has the necessary substrate to generate seizures.

Proteasome inhibitor attenuates skeletal muscle reperfusion injury by blocking the pathway of nuclear factor-kappaB activation.

BACKGROUND: Nuclear factor-kappaB is a key transcriptional factor in the regulation of inflammatory factors that are involved in tissue reperfusion injury, but conflicting data have been presented in the literature. The proteasome regulates proteins that control cell-cycle progression and apoptosis, and inhibition of the proteasome has been shown to reduce nuclear factor-kappaB activation and reperfusion injury. Although bortezomib is a potent proteasome inhibitor, its role in skeletal muscle reperfusion injury has not been documented, and its effects on the regulation of inflammatory factors in reperfused tissue are unclear. In this study, the authors investigated the role of nuclear factor-kappaB in skeletal muscle reperfusion injury and the effect of bortezomib (a proteasome inhibitor) on reperfusion injury. METHODS: Pedicled cremaster muscle flaps from bortezomib-treated and phosphate-buffered saline-treated control mice were subjected to 4.5 hours of ischemia and 90 minutes of reperfusion. RESULTS: During reperfusion, arterial diameters and blood flow recovered earlier and more completely in bortezomib-treated muscle than in controls. Compared with controls, Western blot analysis demonstrated a significant reduction in degradation of nuclear factor-kappaB inhibitory protein and expression of inducible nitric oxide synthase protein in bortezomib-treated muscle at the end of reperfusion. Immunohistochemistry showed decreased nuclear factor-kappaB p65-binding activity and down-regulated protein expression of intercellular adhesion molecule-1 and nitrotyrosine, accompanied by less muscle edema and inflammation as proven by histologic examination. CONCLUSIONS: Bortezomib effectively blocks nuclear factor-kappaB activation in attenuating muscle reperfusion injury through inhibiting nuclear factor-kappaB inhibitory protein degradation. Therefore, inhibition of proteasome activity may provide a novel therapeutic strategy for the treatment of skeletal muscle reperfusion injury.

The synergistic action of a VEGF-receptor tyrosine-kinase inhibitor and a sensitizing PDGF-receptor blocker depends upon the stage of vascular maturation.

OBJECTIVE: To investigate the effects of tyrosine-kinase inhibitors of vascular endothelial growth factor (VECF) and platelet-derived growth factor (PDCF)-receptors on non-malignant tissue and whether they depend upon the stage of vascular maturation. MATERIALS AND METHODS: PTK787/ZK222584 and CGP53716 (VEGF- and PDGF-receptor inhibitor respectively), both alone and combined, were applied on chicken chorioallantoic membrane (CAM). RESULTS: On embryonic day of CAM development (E)8, only immature microvessels, which lack coverage of pericytes, are present: whereas the microvessels on E12 have pericytic coverage. This development was reflected in the expression levels of pericytic markers (alpha-smooth muscle actin, PDGF-receptor beta and desmin), which were found by immunoblotting to progressively increase between E8 and E12. Monotherapy with 2 microg of PTK787/ZK222584 induced significant vasodegeneration on E8, but not on E12. Monotherapy with CGP53716 affected only pericytes. When CGP53716 was applied prior to treatment with 2 microg of PTK787/ZK222584, vasodegeneration occurred also on E12. The combined treatment increased the apoptotic rate. as evidenced by the cDNA levels of caspase-9 and the TUNEL-assay. CONCLUSION: Anti-angiogenic treatment strategies for non-neoplastic disorders should aim to interfere with the maturation stage of the target vessels: monotherapy with VEGF-receptor inhibitor for immature vessels, and combined anti-angiogenic treatment for well developed mature vasculature.

Variable pressure and environmental scanning electron microscopy: imaging of biological samples.

The use of elevated gas pressures in the sample chamber of a scanning electron microscope (i.e., variable pressure SEM, or VPSEM) together with specialized electron detectors create imaging conditions that allow biological samples to be examined without any preparation. Specific operating conditions of elevated pressures combined with sample cooling (usually restricted to the environmental SEM range) can allow hydrated samples to be maintained in a pristine state for long periods of time. Dynamic processes also can be easily observed. A wider range of detector options and imaging parameters introduce greater complexity to the VPSEM operation than is present in routine SEM. The current instrumentation with field emission electron sources has nanometer-scale beam resolution (approx 1 nm) and low-voltage beam capability (0.1 kV). However, under the more extreme variable pressure conditions, useful biological sample information can be achieved by skilled operators at image resolutions to 2 to 4 nm and with primary electron beam voltages down to 1.0 kV. Imaging relating to electron charge behavior in some biological samples, generally referred to as charge contrast imaging, provides information unique to this VPSEM and environmental SEM that closely relates to luminescence imaged by confocal microscopy.

Transpupillary thermotherapy: effect of wavelength on normal primate retina.

PURPOSE: To correlate changes in primate fundus after transpupillary thermotherapy (TTT) at two wavelengths. METHODS: Twelve primate eyes were treated with TTT using a wavelength of 635 nm (n=7) or 810 nm (n=5). Laser parameters were as follows: 635 nm (spot size, 1 mm; duration, 30-8 seconds; and fluence [power over time], 20-91.4 J/cm) and 810 nm (spot size, 2 mm; duration, 60 seconds; and fluence, 96-436 J/cm). Fundus photography, fluorescein and indocyanine green angiography, and enucleation were performed at time 0 or 2 weeks after TTT for histologic analysis. RESULTS: Threshold for fundus lesions (91.4 J/cm at 635 nm and 191 J/cm at 810 nm), acute and chronic retinal damage shown by histologic analysis (79.2 J/cm at 635 nm and 96 J/cm at 810 nm), and choroidal vessel occlusion (50 J/cm at 635 nm and 96 J/cm at 810 nm) were lower at 635 nm. Disorganization of the retina and retinal pigment epithelium was seen for both wavelengths at time 0 and 2 weeks after TTT. Occlusion of the choriocapillaris and choroidal stromal vessels was noted only in specimens obtained 2 weeks after TTT. CONCLUSIONS: TTT resulted in acute and delayed damage to the neurosensory retina that persisted at 2 weeks. The 635-nm wavelength demonstrated a lower threshold fluence for visible fundus lesions, retinal damage, and choroidal vascular occlusion than the 810-nm laser.

Fungal polysaccharopeptide inhibits tumor angiogenesis and tumor growth in mice.

Angiogenesis is crucial to tumor growth and metastasis, and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. The present study has made use of the S180 tumor-bearing mouse model to investigate the polysaccharopeptide, PSP, isolated from the edible mushroom Coriolus versicolor, a herbal medicine known for its anti-angiogenesis properties. Quantitative analysis of microcorrosion casting of the tumor tissue showed more angiogenic features such as dense sinusoids and hot spots, in control (untreated) than in PSP-treated animals. Immunostaining of tumor tissues with antibody against the endothelial cell marker (Factor VIII) demonstrated a positive correlation in that both the vascular density and tumor weight were lower in mice treated with PSP. Morphometric analysis of corrosion casts revealed that, even though the total amount of new vessel production was reduced, the basic tumor type-specific vascular architecture was retained. However, the expression of vascular endothelial cell growth factor (VEGF) in these tumors was suppressed. In conclusion, anti-angiogenesis should be one of the pathways through which PSP mediated its anti-tumor activity.

Directly applied low intensity direct electric current enhances peripheral nerve regeneration in rats.

The influence of direct electric stimulation on nerve regeneration was studied in a model of crush injury of the sciatic nerve of rats. Forty-three rats were used and distributed in four groups according to the procedure: (1) intact nerve, inactive circuit; (2) crush injury, inactive circuit; (3) intact nerve, active circuit; (4) crush injury, active circuit. The low intensity continuous current circuit (1 microA) was implanted in the lumbar region, the anode being fixed to the muscles proximally and the cathode below the nerve distally to the lesion site. The Sciatic Functional Index (SFI) was evaluated at weekly intervals for 3 weeks, the sciatic nerve being resected on the 21st day for histologic and morphometric studies. The SFI progressively improved and the average fiber nerve density recovered to a nearly normal value in Group 2 and increased in Group 4 compared with the control groups (1 and 3), but this was accompanied by a decreased average fiber nerve diameter. Both number and diameter of inter and intra-fascicular blood vessels increased in the stimulated nerves. We conclude that low intensity direct electric stimulation enhances nerve regeneration following a controlled nerve crush injury and increases blood supply by increasing number and diameter of vasa nervorum.

Distribution of various types of oligodendrocytes and cellular localisation of iron in the frontal cortex of the adult rat.

Oligodendrocytes, called oligodendroglia, are present in the white and grey substance of the CNS. They constitute a heterogeneous group, in which 3 types of these cells have been distinguished: those of light, medium and dark cytoplasm. They locate themselves at the neurones and blood vessels. The number of light oligodendrocytes decrease with age, while the number of dark oligodendrocytes increase. In histochemical studies the product of the reaction located itself in the body and processes, indicating the participation of these cells in the metabolism of iron.

Beneficial effects of L-arginine supplementation in experimental hyperlipemia-hyperglycemia in the hamster.

The objective of this study was to evaluate whether administration of L-arginine, the substrate for nitric oxide synthesis, was able to ameliorate the endothelial dysfunction and the morphological changes induced by the combined insult of hyperlipemia and hyperglycemia. To this purpose, golden Syrian hamsters were rendered simultaneously hyperlipemic and diabetic (HD group) for 24 weeks, and then orally treated with 622.14 mg/kg per day L-arginine, for 12 weeks (HD + L-arg group). The following assays were carried out: (1) spectrophotometric: concentrations of circulating glucose, cholesterol, and creatinine, the activity of angiotensin-converting enzyme (ACE), and the osmotic fragility of erythrocyte plasmalemma; (2) myographic: the endothelium-dependent and -independent relaxation of the resistance arteries (i.d. 210-250 microm) to 10(-8) to 10(-4) M acetylcholine (ACh) or sodium nitroprusside (SNP); and (3) electron-microscopic: the ultrastructure of the resistance arteries, myocardium, and kidney glomeruli, which are main targets of hypertensive complications. The results showed that oral supplementation with L-arginine in simultaneous hyperlipemia-hyperglycemia induced in hamsters had favorable effects on: (1) homeostasis, i.e., diminished the concentration of circulating glucose (by ~63%) and cholesterol (by approximately 10%), reduced the ACE activity (by approximately 45%), and lowered the osmotic fragility of erythrocyte plasmalemma (as marker for the oxidative stress in plasma); (2) mesenteric resistance arteries, which showed (in 10(-4) M ACh) an improved endothelium-dependent relaxation (72.40+/-4.6% in the HD + L-arg group vs 61.90+/-1.45% in the HD group) and a reduced thickness (approximately 1.32-fold) of the smooth muscle cells' extracellular matrix; and (3) the heart, which displayed approximately 16% diminishing of the thickness of the left ventricular wall, and an apparently normal structure of the myocardium; the restoration of the thickness of the pericapillary extracellular matrix to almost normal dimensions was also observed. Administration of L-arginine did not modify the high level of plasma creatinine determined for the HD group (approximately 48% increased vs control group) and had no effect on the thickened, nodular basal lamina of the kidney capillaries. The results indicate that endothelial dysfunction established in combined hyperlipemia-diabetes is distinctive for each vascular bed (mesenteric arterioles, heart capillaries, kidney glomerular capillaries), and there is a reversible stage of the dysfunction in which L-arginine oral supplementation induced beneficial effects.

Degranulation of mast cells in the rat thalamus.

Brain mast cells are selectively concentrated in the thalamus of many mammalian species. We here describe by light and electron microscopy in the normal thalamus of adult rats the features of mast cell degranulation, which indicate an active release of the mediators stored in their intracellular granules. The state of activity of thalamic mast cells in basal conditions was found to range from the release of a few granules to a massive degranulation, and the latter process was much less frequent than a partial degranulation. Mast cells were subdivided in three categories (fully granulated, partially or massively degranulated) on the basis of their cytoplasmic features revealed by acidic toluidine blue staining; the fully granulated cells were found to represent only 23 % of thalamic mast cells. This strategy of evaluation could be of help in the comparison of the functional correlates of mast cells in different conditions and experimental paradigms. However, we also demonstrated with image analysis a continuum of the variation of staining intensity of granulated and degranulating mast cells, without a sharp subdivision into different categories. Therefore our results reveal that the vast majority of mast cells are active in the thalamus in basal conditions, and that image analysis can provide an objective index of the activity of these cells.

Detailed examination of vascular lesions triggered by an inhibitor of endothelium-derived relaxing factor.

BACKGROUND: Inhibition of an endothelium-derived relaxing factor (EDRF) may contribute to the pathogenesis of thrombotic arterial occlusions. EXPERIMENTAL DESIGN: We measured the blood pressure and urinary excretion of protein, sodium, and potassium and histologically examined the brains, hearts, and kidneys in normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) fed on a diet containing: (a) EDRF inhibitor (L-N-nitroarginine:L-NNA); (b) L-arginine, which reverses the effect of L-NNA; or (c) both L-NNA and L-arginine for 1 to 8 weeks. In addition, we examined L-NNA-treated SHRSP, the blood pressures of which were lowered using hydralazine. Furthermore, we produced and examined Goldblatt's renal hypertensive rats, which are of a different type from those resulting from the L-NNA treatment. RESULTS: Both WKY and SHRSP rats fed on a diet containing L-NNA suffered from hypertension and cerebral infarctions in a dose-dependent manner. Cerebral infarctions occurred whether or not SHRSP rats were treated with an antihypertensive agent when they were fed a high dosage of L-NNA. In contrast, SHRSP rats, treated simultaneously with both L-NNA and L-arginine, suffered few cerebral infarctions, although they were severely hypertensive. In addition, there were no cerebral infarctions in Goldblatt's renal hypertensive rats, although they suffered from advanced hypertension. CONCLUSIONS: The data indicate that the inhibition of EDRF injures the vessel walls and encourages platelet adhesion to the damaged areas. The adhering platelets narrow the lumen with resultant thrombotic arterial occlusions. Pathophysiologic conditions that decrease EDRF synthesis appear to play an important role in cerebral, renal, and myocardial infarctions.

Regulation of angiogenesis in vitro by collagen metabolism.

The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ecstatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis-hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by adding L-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix.

Morphological evaluation of the atherogenicity of corn oil, hydrogenated corn oil, and butter fat in quail.

The atherogenicity of corn oil, hydrogenated corn oil, and butter fat was studied using 57 fourty-day-old male Japanese quails. The animals were fed one of the following diets: basal diet, basal diet with corn oil plus cholesterol, basal diet with hydrogenated corn oil plus cholesterol, or basal diet with butter fat plus cholesterol. Each atherogenic diet contained 15% fat and 2% cholesterol. Marked hypercholesterolemia developed in all fat-fed groups after 3 weeks or 3 months, but no significant difference was seen among the groups. The degree of the luminal narrowing of the ascending aorta and brachiocephalic arteries was highest in the corn oil group and lowest in the hydrogenated fat group. Ultrastructurally, all the fat-fed groups showed similar cellular changes in their aortic lesions. The major cell type of the thickened intima was fibroblast-like cells with or without lipid droplets. Immunohistochemical studies disclosed that alpha-1-antichymotrypsin was strongly positive for fibroblast-like cells in the thickened intima, whereas it was negative for those in the tunica media of the ascending aorta and its branches. These data suggest that hydrogenated corn oil has a less potent stimulating effect on the thickening of the arterial intima than corn oil and butter fat.