Trafficking in blood vessel development

Angiogenesis - Blood vessels demonstrate a multitude of complex signaling programs that work in concert to produce functional vasculature networks during development. A known, but less widely...     

Androgen receptors in thymic epithelium modulate thymus size and thymocyte development

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.     

Molecular mechanisms of blood vessel growth

The basic molecular mechanisms governing how endothelial cells, periendothelial cells and matrix molecules interact with each other and with numerous growth factors and receptors, to form blood vessels have been presented. The many insights gained from this basic knowledge are being extended to further understand pathological angiogenesis associated with disorders such as arterial stenosis, myocardial ischemia, atherosclerosis, allograft transplant stenosis. wound healing and tissue repair. As a result, novel angiogenic and anti-angiogenic molecules are rapid-ly entering the clinic, with the promise of relief from a host of medical disorders.     

血管异位钙化的细胞生物学机制

钙化是动脉粥样硬化的一个显著特征 ,以往认为动脉钙化是动脉粥样硬化发生后的一个退行性过程 ,但是由于在钙化的动脉及瓣膜发现了作为羟基磷灰石发生中心的基质小泡 (ｍａｔｒｉｘｖｅｓｉｃｌｅｓ ,ＭＶＳ) ,以及一些骨相关蛋白 ,血管的钙化已被认为与骨组织钙化相似 ,是一个主动的调节过程。但是对于这一过程的细胞学机制尚不清楚 ,本文对目前已取得的一些研究进展加以综述。1　基质小泡与钙化　　ＭＶＳ是细胞外的一种 10 0～ 70 0ｎｍ大小的膜包裹的结构 ,骨组织中ＭＶＳ的性质和功能已经有较多的了解 ,70年代以来 ,ＭＶＳ在瓣膜钙化中…     

Magnetic targeting of microspheres in blood flow

Magnetically responsive albumin microspheres can be targeted to the vasculature of specific organs, using extracorporeal magnetic sources. Experiments have been performed on targeting these microspheres to specific regions of normal and tumorous rat tails. This paper quantitatively analyzes the relationship between magnetic forces and the observed microsphere holding. The magnetic forces are determined by the magnetic responsiveness of the microspheres, and by the spatial field of the magnet; both of these are measured. The microsphere holding is defined as that fraction of the microspheres perfusing the tail which are held at a particular site; this is measured at various positions in the tail. The holding as a function of magnetic force is thereby established. To interpret the data, the dynamics of microspheres in blood flow is considered, including motion to a vessel wall, shear forces at the wall, and intersphere attraction. Overall, the method appears favorable for targeting therapeutic drugs to tumor sites in humans.     

An isoform-specific allele of Drosophila N-cadherin disrupts a late step of R7 targeting

Drosophila N-cadherin is required for the formation of precise patterns of connections in the fly brain. Alternative splicing is predicted to give rise to 12 N-cadherin isoforms. We identified an N-cadherin allele, N-cad(18Astop), that eliminates the six isoforms containing alternative exon 18A and demonstrate that it strongly disrupts the connections of R7 photoreceptor neurons. During the first half of pupal development, N-cadherin is required for R7 growth cones to terminate within a temporary target layer in the medulla. N-cadherin isoforms containing exon 18B are sufficient for this initial targeting. By contrast, 18A isoforms are preferentially expressed in R7 during the second half of pupal development and are necessary for R7 to terminate in the appropriate synaptic layer in the medulla neuropil. Transgene rescue experiments suggest that differences in isoform expression, rather than biochemical differences between isoforms, underlie the 18A isoform requirement in R7 neurons.     

VEGF-A,HGF and bFGF are involved in IL-17A-mediated migration and capillary-like vessel formation of vascular endothelial cells

Background: Interleukin (IL)-17A possesses biological activities to promote vascular endothelial cell migration and microvessel development. Objective: To clarify which angiogenic factors are involved in IL-17A-modified angiogenesis-related functions of vascular endothelial cell migration and microtube development or not. Methods: The potential contribution of various angiogenic stimulators to in vitro angiogenic activities of IL-17A was assessed with both modified Boyden Chemotaxicell chamber assay and in vitro angiogenesis assay. Results: The addition of a neutralizing antibody (Ab) for hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-A to the upper and lower compartments in a modified Boyden Chemotaxicell chamber significantly attenuated human dermal microvascular endothelial cell (HMVEC) migration elicited by IL-17A. Moreover, IL-17A-induced capillary-like microvessel development in human umbilical vein endothelial cell (HUVEC) and human dermal fibroblast (HDF) co-culture system was significantly impaired by a neutralizing Ab against HGF, bFGF, VEGF-A, cysteine-x-cysteine ligand 8 (CXCL8)/IL-8 or cysteine-x-cysteine (CXC) chemokine receptor (CXCR)-2. Conclusion: Our findings demonstrate the involvement of HGF, bFGF, VEGF-A and/or CXCL8/IL-8, to various degrees, in migration and microvessel development of vascular endothelial cells mediated by IL-17A.     

Adhesion receptors are differentially expressed on developing thymocytes and epithelium in human thymus.

The thymic microenvironment consists of a network of interrelated cells of epithelial, fibroblastic, endothelial, and hemopoietic origin. Within this environment, the development of specific T-lymphocyte subpopulations partially depends on the selective interaction of T-cell precursors with such cells. Human thymic epithelial cell strains, generated with a defective retroviral vector containing simian virus 40 (SV40) large T antigen and the neomycin resistance gene or by transfection with an SV40 plasmid defective in the origin of replication, provide useful tools for understanding the mechanisms contributing to the control of T-cell maturation. Because interepithelial, epithelial-macrophage, and lymphocyte-epithelial cell interactions are important for thymocyte differentiation, the distribution of integrin and nonintegrin adhesion receptors on these cells and on developing thymocytes in vivo and in vitro has been examined in detail. Our results indicate that the transformed human thymic epithelial cell strains express the common very late antigen (VLA)-beta 1 receptor and unique alpha chains VLA-2, VLA-3, and VLA-6. The cells are also positive for LFA-3 and ICAM-1 and weakly express beta 3, beta 4, and VNR alpha. They do not express the Leu-cellular adhesion molecules (CAM). This phenotypic profile on cultured thymic epithelium generally corresponds to the distribution of integrin and other receptor molecules on thymic epithelial cells in tissue sections. The majority of thymocytes also express the integrin VLA-beta 1 and -beta 2 chains as well as VLA-4, VLA-6, and LFA-1 alpha(L). Three-color flow cytometric analyses show differential levels of expression of these adhesion receptors on human thymocyte subsets. Taken together with the immunohistochemical localization of extracellular matrix molecules, these studies suggest that both the distribution of receptor-ligand pairs and the level of expression of adhesion molecules may influence T-cell development within the thymus.     

Angiogenesis and blood vessel stability in inflammatory arthritis

Objective

To assess blood vessel stability in inflammatory synovial tissue (ST) and to examine neural cell adhesion molecule (NCAM), oxidative DNA damage, and hypoxia in vivo.

Methods

Macroscopic vascularity and ST oxygen levels were determined in vivo in patients with inflammatory arthritis who were undergoing arthroscopy. Vessel maturity/stability was quantified in matched ST samples by dual immunofluorescence staining for factor VIII (FVIII)/α‐smooth muscle actin (α‐SMA). NCAM and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) were examined by immunohistochemistry. Angiogenesis was assessed in vitro, using human dermal endothelial cells (HDECs) in a Matrigel tube formation assay.

Results

A significant number of immature vessels (showing no pericyte recruitment) was observed in tissue from patients with inflammatory arthritis (P < 0.001), in contrast to osteoarthritic and normal tissue, which showed complete recruitment of pericytes. Low in vivo PO ₂ levels in the inflamed joint (median [range] 22.8 [3.2–54.1] mm Hg) were inversely related to increased macroscopic vascularity (P < 0.04) and increased microscopic expression of FVIII and α‐SMA (P < 0.04 and P < 0.03, respectively). A significant proportion of vessels showed focal expression of NCAM and strong nuclear 8‐oxodG expression, implicating a loss of EC–pericyte contact and increased DNA damage, levels of which were inversely associated with low in vivo PO ₂ (P = 0.04 for each comparison). Circulating cells were completely negative for 8‐oxodG. Exposure of HDEC to 3% O₂ (reflecting mean ST in vivo measurements) significantly increased EC tube formation (P < 0.05).

Conclusion

Our findings indicate the presence of unstable vessels in inflamed joints associated with hypoxia, incomplete EC–pericyte interactions, and increased DNA damage. These changes may further contribute to persistent hypoxia in the inflamed joint to further drive this unstable microenvironment.

     

Role of neural guidance signals in blood vessel navigation

Despite the tremendous progress achieved in both vasculogenesis and angiogenesis in the last decade, little is still known about the molecular mechanisms underlying the pathfinding of blood vessels during their formation. However, emerging evidence shows that different axonal guidance cues, including members of the Slit and semaphorin families, are also involved in the blood vessel guidance, suggesting that blood vessels and nerves share common mechanisms in choosing and following specific paths to reach their respective targets. These promising findings open novel avenues not only in vascular biology but also in therapeutic angiogenesis. Indeed, the identification of new molecules involved in the guidance of blood vessels may be helpful in designing angiogenic strategies, which would insure both the formation of new blood vessels and their guidance into an organized and coordinated network.     

Involvement of non-vascular stem cells in blood vessel formation

Blood vessels clearly act as conduits for blood flow, but recently the concept that they are also involved in organ maintenance, especially by providing a niche for organ-specific stem cells, has begun to emerge. Moreover, several lines of evidence suggest that hematopoietic stem cells can differentiate directly into cells composing blood vessels. Recently, cancer stem cells (CSCs) have also been assigned these roles in the cancer microenvironment. Although anti-angiogenic drugs have been developed and are utilized in the clinic for their anti-tumor activity, their suppressive effects on tumor growth have been disappointing. This may be caused by transferring drug resistance from CSCs to endothelial cells. It has been suggested that CSCs localize in the peri-vascular niche. Therefore, it is extremely important to know how the vascular niche maintains CSCs, as such knowledge may enable us to develop promising new approaches to cancer treatment.     

Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo.The seminiferous epithelium is separated by tight junctions between Sertoli cells into a luminal compartment containing spermatocytes and spermatids and a basal compartment containing spermatogonial stem cells (SSCs) and spermatogonia. The basal compartment is bounded above and on the sides by Sertoli cells and below by the basement membrane of the seminiferous tubule and a layer of peritubular myoid (PM) cells. SSCs are thought to reside in a microenvironmental niche in the basal compartment, where extrinsic cues influence their decision to either self-renew or enter the pathway of spermatogonial development (1, 2). They are a minor fraction of the undifferentiated spermatogonia in the basal compartment. The other undifferentiated spermatogonia (progenitors) give rise to differentiating spermatogonia that proliferate mitotically to progress on a developmental pathway toward becoming spermatocytes (3, 4). Our current understanding of the progression of SSCs to differentiating spermatogonia comes mainly from cell kinetic studies, germ cell transplantation assays, and the use of molecular markers that identify different populations of spermatogonia.The leading model for spermatogonial development specifies that when SSCs divide, they either self-renew by becoming two type A-single (A_s) spermatogonia or give rise to type A-paired (A_pr) spermatogonia connected by an intercellular bridge to become undifferentiated spermatogonia (5–7). The pairs continue to divide to form short chains of bridge-connected undifferentiated type A-aligned (A_al) spermatogonia, and these in turn divide to form longer chains of differentiating (type A₁, A₂, A₃, intermediate, and B) spermatogonia.Although SSCs are single cells, not all A_s spermatogonia are likely to be SSCs. There are ∼35,000 A_s spermatogonia in the testes of adult mice (8), but only about 3,000 of these have the ability to regenerate spermatogenesis when transplanted to germ cell-depleted testes (9). Although there are no generally accepted molecular markers specific for SSCs, potential candidates are inhibitor of DNA binding 4 (ID4) and paired box 7 (PAX7), which are expressed in minor subsets of A_s spermatogonia (10–12). However, it remains to be reported if ID4 and PAX7 are coexpressed in the same subset of A_s spermatogonia. SSCs also share molecular markers with undifferentiated spermatogonia, including Nanos2, Gfra1, Zbtb16, Bcl6b, and THY1(13–17). In addition, differentiating spermatogonia have characteristic molecular markers, including Ngn3, Nanos3, Spo11, and KIT (18–22). These molecular markers have been proven to be valuable tools for monitoring the presence or absence of different populations of spermatogonia.A conditional knockout (cKO) of the androgen receptor (Ar) gene in PM cells resulted in progressive loss of spermatogonia beginning at postnatal d 21, leading to disorganization of the seminiferous epithelium and infertility (23). This strongly suggested that androgens regulate genes in PM cells whose products are essential for SSC maintenance. In other studies, mice heterozygous for a global mutation in the gene for glial cell-derived neurotrophic factor (Gdnf) had reduced stem cell reserves, whereas mice with a transgene overexpressing GDNF experienced an overaccumulation of undifferentiated spermatogonia (24). GDNF also was reported to be critical for development of SSCs in vitro and their ability to restore spermatogenesis after transplantation to the testes of germ-cell-depleted mice (25, 26). This led us to hypothesize that regulation of GDNF production in PM cells by testosterone (T) is essential for SSC maintenance. In studies to test this hypothesis, we determined that PM cells isolated from adult mice and treated in vitro with T produce GDNF but not when untreated. We also found that SSCs from neonatal mice co-cultured with T-treated PM cells and transplanted to testes of germ cell-depleted mice restored spermatogenesis but not when they were co-cultured with untreated PM cells (10). These results supported the hypothesis but did not rule out the possibilities that PM cells or Sertoli cells produce other factors in vivo in addition to GDNF that are responsible for SSC self-renewal and differentiation. In these studies, we generated mice with a cKO of the Gdnf gene in PM cells to test the hypothesis that the production of GDNF by PM cells is essential for the in vivo development of undifferentiated spermatogonia.