Fine structure of cellular and vascular reaction in brain tuberculomas: a model for phagocytosis in the CNS

The fine structure of cells of the mononuclear phagocyte series (MPS) and a few other cells with phagocytic capacity, has been critically evaluated, mainly from an electronmicroscopic examination of the reactive border zone of 11 human brain tuberculomas, which provide ideal material for the study of macrophages. Most of them appeared to be blood monocyte-derived epithelioid cells of various forms and stages. The cytoplasm of these cells showed either more rough ER representing protein synthesising activity; or more frequently, phagosomes, phagolysosomes, dense bodies or empty vacuoles, representing various stages of ingestion and digestion of necrotic material. Often such material, which was more or less osmiophilic, was seen abundantly between the cells. These actively phagocytic cells occasionally undergoing, mitosis, are referred to as "epithelioid macrophages" and were morphologically similar to the "activated microglia" described in other conditions. They also showed a tendency to be closely adjacent to each other and occasionally fuse to form giant cells. There were also a number of lymphocytes and plasma cells. The latter showed various stages of active and granular or depleted and distended rough ER tubules, phagocytic activity and tendency to fuse. Expected vasculitis and small vessel necrosis formed part of this granulomatous reaction. Constituents of oedematous or necrosed brain tissue were seen immediately around the reactive zone of these tuberculomas, the most frequent being reactive astrocytes, many of which showed membrane-bound vacuoles. It is conceivable that the excessive pleomorphic cellular, vascular and necrotic reaction in these brain tuberculomas could have resulted from a delayed type of hypersensitivity to a very small quentity of antigenic tuberculoprotein, which probably initiates the chain of immunologic responses.     

Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors

Often described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered >99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1- micro m confocal optical sections, were <2.5 micro m in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors.     

Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.     

Foxc1 is required for early stage telencephalic vascular development

Background : The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon. Results: The Foxc1 loss of function mutant mouse, Foxc1^ch/ch, showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites. Conclusions: These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon. Developmental Dynamics 244:703–711, 2015. © 2015 Wiley Periodicals, Inc.     

Angiosarcomas arising in edematous extremities: immunostaining for factor VIII-related antigen and ultrastructural features

Immunostaining for Factor VIII-related antigen was seen in deparaffinized sections from 19 of 20 postmastectomy angiosarcomas and from four of four sarcomas that arose in chronically edematous tissue unrelated to breast carcinoma. Staining was also seen in sections from two malignant hemangioendotheliomas, four capillary hemangiomas, and one granulation tissue specimen. Sections from two lymphangiomas were immunonegative for Factor VIII-related antigen in the endothelium of lymphatic channels, whereas staining was observed in the surrounding normal blood vessels. Electron microscopic study of four postmastectomy angiosarcomas disclosed ultrastructural features (fenestrae, intense pinocytotic activity, cell junctions, and Weibel-Palade bodies) supporting the blood vascular endothelial nature of the neoplastic cells. It is concluded that a neoplastic blood vessel component is present in sarcomas that arise in chronically edematous tissues. It is questionable whether a lymphatic component is also present. These tumors, therefore, should be regarded as angiosarcomas rather than lymphangiosarcomas.     

15例血管外皮瘤的临床病理及免疫组化观察

通过光镜、网织纤维及免疫组化染色对15例血管外皮瘤进行了研究,其诊断要点为:(1)血管极为丰富,遍布整个肿瘤组织,而呈非灶性区域性的血管外皮瘤样区,血管腔隙大小不等、形状不一,从大的扩张薄壁血窦、鹿角状血管,到中等大小直至小的毛细血管均可见到;(2)网织纤维极为丰富,遍布全瘤,且分布规律,总在血管基底膜外围绕单个或2～3个瘤细胞呈细网状,瘤细胞巢内均有网织纤维;(3)免疫组化染色瘤细胞仅对Vimentin呈阳性反应。本文还讨论了与滑膜肉瘤等的鉴别诊断。     

Morphological aspects of angiogenesis in experimental liver metastases.

In this work we describe the process of angiogenesis in liver metastases of high- and low-metastatic 3LL mouse carcinoma lines. Fourteen days after intrasplenic inoculation of the tumor lines, two types of metastases were observed; a sinusoidal type, containing large convoluted vessels and devoid of immunohistochemically detectable basement membrane, and a portal type, located in the vicinity of portal tracts, characterized by numerous small vessels, and staining positively for basement membrane components. After intrasplenic inoculation of the high-metastatic tumor cells (portal route) only 18.2% of the metastases were portal type, whereas when the tumor cells were injected into the left ventricle (arterial route), a significantly higher percentage of the metastases (33.2%) proved to be portal type. Detailed analysis of the process of angiogenesis were performed only concerning the main, sinusoidal type metastases. After intrasplenic inoculation of tumor cells, vascularization of tumor colonies started on day 6 by the appearance of intratumoral sinusoids lined by endothelial cells. These sinusoids were directly connected with liver sinusoids. Afterward (11 to 14 days), large convoluted vessels developed within the metastases, in which tumor globules protruded. These globules were covered by factor VIII-related antigen-positive endothelial cells. The functioning vascular nature of these vessels were proven by supravital staining with Hoechst 33342 dye and by bromodeoxyuridine labeling. The first event of the angiogenesis in sinusoids and veins seemed to be the separation of the endothelial cells from their basement membrane, demonstrated by electron microscopic immunohistochemistry (laminin, fibronectin). This process elicited vigorous proliferation of the matrix-deprived endothelial cells, shown by the increased bromodeoxyuridine labeling index and by the increased number of endothelial cell nuclei per mm vessel length. Morphometric analysis of the sinusoids in the perimetastatic zone (up to 100 mu) and in the normal liver parenchyma showed neither dilatation of the vessels nor sprouting of new vessels in the former region. There was no difference in the neovascularization of the liver metastases of the high- and low-metastatic carcinoma lines. The dominant type of angiogenesis in liver metastases can be determined by the unique basement membrane architecture of the liver and by the high affinity of 3LL tumor cells to the endothelial side of basement membrane during invasion.     

Characterization of the Hemorrhagic Reaction Caused by Vibrio vulnificus Metalloprotease, a Member of the Thermolysin Family

Vibrio vulnificus is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (V. vulnificus protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.     

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.     

The structure of the developing blood vessels of the neocortical anlage of the human embryo

Using light and electron microscopy the structure of blood vessels of neocortical anlage of human 7-12 embryos was studied. It was shown that at the early stage of formation of intraorgan vascular network the wall of blood vessels of ventricular zone successively differentiate, which is characterized by the appearance of second layer of cells (pericytes), accumulation of basement membrane components, widening of the zone of contacts between endotheliocytes and establishment of the contacts with bipolar cells of neocortex anlage. The morphological data obtained assist in comprehension of physiological aspects of formation of blood brain barrier and regulation of blood flow in human embryonal neocortex.     

New vessel formation after surgical brain injury in the rat's cerebral cortex II. Formation of the blood vessels distal to the surgical injury

We investigated the features of newly formed blood vessels after surgical brain injury of the rat's cerebral cortex distal to the operated region. We document the process of split mature blood vessels by an endothelial bridge and morphological features of newly formed vessels. We did not observe a disruption of brain parenchyma. The endothelial lining in vessels was complete. The morphological features of the endothelial cells and basement membrane show that non-sprouting angiogenesis takes place distally to the surgical injury.     

Vasculitis in lichen sclerosus: an under recognized feature?

AIMS: To analyse 90 vulvar and 72 penile cases of lichen sclerosus (LS) on haematoxylin and eosin sections for vascular changes and the vascular infiltrates immunohistochemically with antibodies to T cells, B cells and antigen-presenting dendritic cells. LS is a skin disease of presumed autoimmune origin. Autoimmune diseases are mediated by lymphocytes which occasionally produce a lymphocytic vasculitis. METHODS AND RESULTS: Three types of lymphocytic infiltrates were identified: (i) perivascular lymphocytic infiltrates without damage to vessel walls; (ii) lymphocytic vasculitis in three forms: (a) concentric lymphohistiocytic infiltrates with lamination of the adventitia by basement membrane material which was typical for penile LS; (b) lymphocytic vasculitis with dense perivascular lymphocytic cuffing with occasional fibrin deposition in vessel walls and subendothelial lymphocyte infiltration, quite common in vulvar LS; and (c) intramural lymphocytic infiltrates in large muscular vessels; (iii) leukocytoclastic vasculitis in LS was exceptionally rare. In lymphocytic vasculitis, CD20+ B cells, CD4+ T cells and dendritic cells were the principal infiltrating cells. CONCLUSIONS: Dendritic cells capture (foreign) antigens after entry into the affected tissues and initiate immune responses acting as a matrix on which antigen-specific T and B cells interact. The described vascular features are indicative of antigen-mediated vasculitic changes in LS.     

Alterations in rat's brain capillaries in a model of focal cerebral necrosis.

Focal brain compression causes cerebral tissue damage. In this study we followed alterations in capillary ultrastructure in the rat cortex and neurohypophysis caused by 40 mm Hg compression for 15 minutes. One day after experiment we observed clogging of capillaries, accumulation of collagen fibrills under the basement membrane and necrosis or apoptosis of endothelial cells. Four days after it the basement membrane was multiplicated, blurred and thickened. In the neurohypophysis the formation of vessels lined with the atypical continuous endothelium was seen. There was also evidence for the migration of pericytes through the blurred basement membrane and the differentiation of pericytes into endothelial cells. Thus, vascular injury in the compressed brain is followed by a highly ordered sequence of processes in the basement membrane and perivascular cells leading to capillary repair.     

大鼠松果体血脑屏障的电镜细胞化学实验观察

目的：观察SD大鼠松果体血脑屏蔽的超微结构。方法：采用硝酸镧示踪电匀细胞化学结合常规电镜技术观察SD大鼠松果体毛细血管的超微结构。结果：（1）松果体毛细血管为有孔型,未见紧密连接,内皮细胞外围有周细胞,并存在较宽的,疏松的絮状基膜样物质,其中散在分布松果体细胞及神经胶质细胞的突起。（2）硝酸镧颗粒广泛分布在毛细血管腔内及其周期基膜样物质中,血管周隙处也有颗粒分布,而内皮细胞,周细胞及其突起内未见颗粒分布,结论：SD大鼠松果体缺乏血脑屏蔽结构,大分子物质容易通过毛细血管进入血管周隙。     

Blood vessels and tissue space associated with the brain of the rat

Tissues from adult Sprague-Dawley rats fixed by perfusion with buffered aldehydes for a combined study of the vascular system of the brain are described in light and electron microscopy. In these preparations lack of shrinkage prevents the formation of perineuronal and perivascular spaces. However, connective tissue stains indicate restricted tissue space along the course of small arteries and veins. In fine structure this space is found within the walls of the vessels. It consists of a tubular extension of tissue space bounded inwardly by the endothelial boundary (basement) membrane and outwardly by the neural boundary membrane. Between these boundaries the formed elements of the media and the adventitia are found. The media consists of a thin layer of smooth muscle cells; each cells being enclosed in its own boundary membrane. The adventitia consists of cells and fibrous elements of the connective tissues which are derived, near the surface of the brain, from the intermingling of pial and vascular leptomeninges. This “neural” portion of the tissue space extends from the depths of the capillary bed (where it is obliterated by the fusion of boundary membranes), along the course of the blood vessels, through the subarachnoid space and into the general tissue space of the body.     

Electron microscopical studies of vessels in diabetic peripheral neuropathy.

the results of an electron microscopical study of sural nerve biopsies from 11 patients with diabetic neuropathy are presented. Thrombi were seen in six cases in at least one intraneural vessel; nine cases showed hyperplasia of endothelial cells, and in seven out of these nine the hyperplasia was sufficient to occlude completely the lumen of small vessels; six cases showed degenerate pericytes and endothelial cells, and in some cases endothelial cells had been shed from the vessel wall, exposing the blood within the vessel to the underlying basement membrane; in five cases large lipid droplets were seen within endothelial cells. Abnormalities of the vessel wall would result in decreased fibrinolytic activity and a reduction of the antiplatelet aggregating proprties of the vessel. Desquamation of endothelial cells from the vessel wall, with exposure of platelets to underlying collagen, may act as a trigger for thrombus formation, particularly as the blood of diabetic patients is often in a hypercoagulable state. The significance of hyperplasia of endothelial cells is at present unknown but, once established, this too would result in profound alterations of loal blood flow and ischaemia of nerve. Damage to endothelial cells may also allow seepage of haematological constituents into the vessel wall, resulting in its progressive thickening.     

Three-dimensional cytoarchitecture of angiogenic blood vessels in a gelatin sheet implanted in the rat skeletal muscular layers

To demonstrate the structure of angiogenic blood vessels three-dimensionally, a gelatin sponge sheet immersed in a vascular endothelial growth factor (VEGF) solution was implanted in the rat dorsal muscular layer, and examined by light microscopy and scanning electron microscopy (SEM) 5 days to 2 weeks after implantation. Light microscopy of anti-collagen IV antibody immunostained specimens enabled a determination of the basement membrane tube of newly formed blood vessels in the implanted sponge sheet. The tubes were 5-40 microm in diameter, and sometimes tapered to a slender cord within the vascular network. The SEM study of 30% KOH treated tissues revealed two types of tapering ends of newly formed blood vessels. One consisted of endothelial cells with microprojections, and lacked any investment of pericytes over the length of 5-20 microm. The other type was a tapering tip of the endothelial tube covered with pericytic processes. The presence of long processes of pericytes extending beyond the tip of the endothelial tube and connecting to the adjacent vessel wall indicates that this type was produced by endothelial tube regression. Thus, the present study supports the ideas that endothelial tube formation is followed by pericyte coverage at the sprouting tip, and that endothelial tube regression precedes pericyte detachment at the regressing site.     

小鼠血脑屏障的组织发生

探讨脑内毛细管的生长发育与形态结构和功能的关系。方法：用透射电镜对３５只不同发育时期小鼠血脑屏障建立过程中的超微结构及血管密度进行了分析，结果：胚胎第１５、１８天血管密度较低，出生后第一天血管度增长明显，第７天下降，以后随年龄增长管密度逐渐增加。     

Diabetic neuropathy: a clinical and pathological study of 8 cases]

Eight cases of diabetic neuropathy are reported, including 5 cases of mixed type sensori-motor-autonomic neuropathy and 3 cases of mononeuropathy multiplex. Sural nerve examination was made in 8 cases. Pathological study showed not only demyelination of the myelinated fibres, but also axonal degeneration. There was different degree of thickening of vascular wall and stenosis of vascular lumen in these 8 cases, one of which showed thrombosis of small vessels in between the nerve fascicles. Marked thickening of basement membrane of the endothelial cells of the blood vessel and of the Schwann's cells was identified by electron-microscopy. The muscles in 5 cases showed neurogenic changes, and one of these 5 cases exhibited syndrome of diabetic amyotrophy. The pathological changes, clinicopathological classification and pathogenesis of the disease are discussed.