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Objective: To develop a reliable method for the isolation and longterm culture of microvessel endothelial cells from human endometrium and to evaluate their response to angiogenic growth factors and steroid hormones in comparison to endothelial cells derived from other organs. Methods: Endometrial tissue from hysterectomy specimens were digested sequentially with collagenase and trypsin, cultured for 24 h, then selected by adhesion to anti-CD-34 coated magnetic beads. Alternatively, anti-CD-34-coated beads could also be substituted by Ulex europaeus agglutinin-1, anti-PECAM, or anti-E-selectin-coated beads. Characterization of the isolated cultures included expression of endothelial cell markers, regulation of E-selectin in response to TNF-α, proliferative response to angiogenic growth factors, and expression of progesterone and estrogen receptors. We also analyzed the relative binding affinity of VEGF on endometrial endothelial cells in comparison to other endothelial cell types. Results: Selection on anti-CD-34-coated beads eliminated contaminating cells and resulted in a homogeneous population of human endometrial endothelial cells (HEEC), as assessed by expression of PECAM, von Willebrand's factor, and uptake of acetylated-LDL. HEEC also upregulated E-selectin in response to TNF-α in a manner similar to that seen for other endothelial cell types. Expression of progesterone and estrogen receptor was revealed by immunocytochemistry and RT-PCR consistently until passage 5. Endometrial endothelial cells were more responsive to growth stimulation by VEGF than were dermal endothelial cells isolated under similar conditions. Further characterization indicated that VEGF bound more avidly to HEEC than to other endothelial cell types. Conclusions: Human endometrial endothelial cells were isolated to homogeneity by a two-part protocol and successfully passaged under culture conditions similar to those used for other endothelial cell types. The HEEC were... [ABSTRACT FROM AUTHOR]

Shear stress is an important physical factor that regulates proliferation, migration, and morphogenesis. In particular, the homeostasis of blood vessels is dependent on shear stress. To mimic this process ex vivo, efforts have been made to seed scaffolds with vascular and other cell types in the presence of growth factors and under pulsatile flow conditions. However, the resulting bioreactors lack information on shear stress and flow distributions within the scaffold. Consequently, it is difficult to interpret the effects of shear stress on cell function. Such knowledge would enable researchers to improve upon cell culture protocols. Recent work has focused on optimizing the microstructural parameters of the scaffold to fine tune the shear stress. In this study, we have adopted a different approach whereby flows are redirected throughout the bioreactor along channels patterned in the porous scaffold to yield shear stress distributions that are optimized for uniformity centered on a target value. A topology optimization algorithm coupled to computational fluid dynamics simulations was devised to this end. The channel topology in the porous scaffold was varied using a combination of genetic algorithm and fuzzy logic. The method is validated by experiments using magnetic resonance imaging readouts of the flow field., (Copyright © 2012 Wiley Periodicals, Inc.)

Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion. In vitro, enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the in vivo regulation of metalloproteinase-9 activation and VEGF signaling.

Regeneration of the endometrium after menstruation requires a rapid and highly organized vascular response. Potential regulators of this process include members of the vascular endothelial growth factor (VEGF) family of proteins and their receptors. Although VEGF expression has been detected in the endometrium, the relationship between VEGF production, receptor activation, and endothelial cell proliferation during the endometrial cycle is poorly understood. To better ascertain the relevance of VEGF family members during postmenstrual repair, we have evaluated ligands, receptors, and activity by receptor phosphorylation in human endometrium throughout the menstrual cycle. We found that VEGF is significantly increased at the onset of menstruation, a result of the additive effects of hypoxia, transforming growth factor-alpha, and interleukin-1beta. Both VEGF receptors, FLT-1 and KDR, followed a similar pattern. However, functional activity of KDR, as determined by phosphorylation studies, revealed activation in the late menstrual and early proliferative phases. The degree of KDR phosphorylation was inversely correlated with the presence of sFLT-1. Endothelial cell proliferation analysis in endometrium showed a peak during the late menstrual and early proliferative phases in concert with the presence of VEGF, VEGF receptor phosphorylation, and decrease of sFLT-1. Together, these results suggest that VEGF receptor activation and the subsequent modulation of sFLT-1 in the late menstrual phase likely contributes to the onset of angiogenesis and endothelial repair in the human endometrium.

METH-1/ADAMTS1 is a member of a newly described family of genes that contain metalloprotease, disintegrin, and thrombospondin-like motifs. We have recently shown that METH-1 protein is a potent inhibitor of angiogenesis. Here, we demonstrate that secreted human pro-METH-1 is processed in two consecutive steps to release both p87 and p65 active forms. The p87 form lacks the N-terminal prodomain and p65 results from an additional processing event in the C-terminal end. Generation of p87 was blocked with specific inhibitors of furin, and incubation of pro-METH-1 with purified furin released the p87 fragment but not p65. Generation of p65 required preformation of p87 and was suppressed by inhibitors of matrix metalloproteases. We demonstrate that matrix metalloproteases 2, 8, and 15 were able to release p65 when p87 was used as substrate. This second processing step removes two thrombospondin repeats from the carboxyl-terminal end of p87-METH-1 and alters the affinity of the protein to heparin and endothelial cultures. Furthermore, this deletion was associated with a reduced activity upon suppression of endothelial cell proliferation. We hypothesize that METH-1 processing is relevant for the modulation of the anti-angiogenic properties displayed by the protein.

All-trans-retinoic acid (RA) and other retinoids modulate cell growth and differentiation, generally favoring terminal cell differentiation and inhibiting carcinogenesis. Retinoids are also reported to inhibit angiogenesis and endothelial cell migration, actions that are also anti-carcinogenic. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine secreted by many tumors. It renders microvessels hyperpermeable to plasma and stimulates endothelial cell migration and division. To investigate further the mechanisms by which RA inhibits angiogenesis, we evaluated the effects of RA on VPF/VEGF-induced angiogenesis and microvascular permeability. RA selectively inhibited the angiogenic response induced by VPF/VEGF, but not that induced by fibroblast growth factor-2 (FGF-2), in the CAM assay. RA and two of its isomers also inhibited the vascular permeabilizing effect of VPF/VEGF but not that induced by histamine. The vascular permeabilization induced by VPF/VEGF and blocked by RA takes place within 1-15 min, too short a time frame for RA to act by modulating transcription through classic retinoid receptors. RA also inhibited VPF/VEGF-induced phosphorylation of PLC-gamma and synthesis of cGMP but actually increased VPF/VEGF binding to cultured endothelial cells. Taken together, these findings indicate that RA selectively blocks VPF/VEGF-induced microvascular permeability and angiogenesis and also identify VPF/VEGF as a major target of RA action. The selectivity of RA's action suggests that other, RA-independent pathways must exist for the angiogenesis induced by FGF-2 and the vascular permeabilizing effect of histamine., (Copyright 2000 Academic Press.)

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.

Progestin-only contraceptives are associated with breakthrough bleeding in up to 50% of users. The causes of blood vessel rupture are not well understood. Here we report that both normal and Norplant-exposed endothelium express progesterone receptor. Experiments performed in vitro on endothelial cells isolated from human endometrium revealed that longterm progesterone exposure leads to suppression of endothelial cell proliferation, inhibition of migration and alteration in the profile of extracellular matrix proteins secreted by human endometrial endothelial cells. In addition, we detected increased levels of matrix metalloproteinase-9 in endothelial cultures treated with progesterone. The effect of progesterone on the cell cycle, along with the increased amounts of matrix-degrading enzymes, could account for breakdown of basement membrane components, vascular fragility and consequent vessel rupture leading to breakthrough endometrial bleeding.

Accumulating evidence suggests that phosphatases play an important role in regulating a variety of signal transduction pathways that have a bearing on cancer. The kinase-associated phosphatase (KAP) is a human dual-specificity protein phosphatase that was identified as a Cdc2- or Cdk2-interacting protein by a yeast two-hybrid screening, yet the biological significance of these interactions remains elusive. We have identified the KAP gene as an overexpressed gene in breast and prostate cancer by using a phosphatase domain-specific differential-display PCR strategy. Here we report that breast and prostate malignancies are associated with high levels of KAP expression. The sublocalization of KAP is variable. In normal cells, KAP is primarily found in the perinuclear region, but in tumor cells, a significant portion of KAP is found in the cytoplasm. Blocking KAP expression by antisense KAP in a tetracycline-regulatable system results in a reduced population of S-phase cells and reduced Cdk2 kinase activity. Furthermore, lowering KAP expression led to inhibition of the transformed phenotype, with reduced anchorage-independent growth and tumorigenic potential in athymic nude mice. These findings suggest that therapeutic intervention might be aimed at repression of KAP gene overexpression in human breast and prostate cancer.

Angiogenesis has been acknowledged as an important requirement for growth and metastasis of tumors. Complete or partial suppression of vascular growth by a number of different strategies has been consistently associated with suppression of tumor expansion and even reduction of tumor burden. Consequently, identification of the molecular pathways of the angiogenic response has been a major focus of interest in academia and industry. The development of tumor-specific anti-angiogenic therapy was also catalyzed by the finding that inhibitors of angiogenesis appeared immune to the development of drug resistance by the tumor cells, a major restrain in current chemotherapy. Although the full identification of players and their cross-talk is still at its infancy, it appears that partial blockade of one of the steps in the angiogenesis cascade, is sufficient to affect capillary morphogenesis. Thus, suppression of specific integrin pathways or vascular endothelial growth factor signaling have been shown effective in the suppression of tumor-mediated angiogenesis and have led to subsequent initiation of clinical trials. In addition to the generation of antibodies or chemical mimetics to interfere with particular steps during vascular organization, several endogenous (or physiological) molecules have also been identified. The list of endogenous modulators of angiogenesis is growing and can offer additional and important tool for the generation of therapies to restrain tumor vascularization. This review will focus one group of such molecules which include the thrombospondins and metallospondins, two families of proteins linked by the presence of a conserved anti-angiogenic functional domain.

Background: Suppression of tumor growth by thrombospondin-1 (TSP-1) has been associated with its ability to inhibit neovascularization. The antiangiogenic activity of TSP-1, as defined by cornea pocket assays, was previously mapped to the amino-terminal portion of the protein within the procollagen region and the type 1 repeats., Methods and Results: We evaluated the specificity and efficacy of different regions of TSP-1 using recombinant fragments of the protein on chorioallantoic membrane (CAM) angiogenesis and endothelial cell proliferation assays. In both assays, fragments containing the second and third type 1 repeats but not the procollagen region inhibited angiogenesis and endothelial cell proliferation. To further define the sequences responsible for the angiostatic effect of TSP-1, we used synthetic peptides. The CAM assay defined 2 sequences that independently suppressed angiogenesis. The amino-terminal end of the type 1 repeats showed higher potency for inhibiting angiogenesis driven by basic fibroblast growth factor (FGF-2), whereas the second region equally blocked angiogenesis driven by either FGF-2 or vascular endothelial growth factor (VEGF). Modifications of the active peptides revealed the specific amino acids required for the inhibitory response. One sequence included the conserved tryptophan residues in the amino-terminal end of the second and third type 1 repeats, and the other involved the amino acids that follow the CSVTCG sequence in the carboxy-terminus of these repeats. Both inhibition in the CAM assay and inhibition of breast tumor xenograft growth in nude mice were independent of the TGF-beta-activating sequence located in the second type 1 repeat., Conclusions: These results indicate that the type 1 repeats of TSP-1 contain 2 subdomains that may independently inhibit neovascularization. They also identify 2 independent pathways by which TSP-1 can block FGF-2 and VEGF angiogenic signals on endothelial cells.

The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis.

The growth of solid tumors has been shown to depend on neovascularization. By understanding the mechanisms that control the neovascular response, it may be possible to design therapeutic strategies to selectively prevent or halt pathologic vascular growth and restrain cancer progression. Thrombospondin-1 is an extracellular matrix protein that among several functions suppresses capillary growth in angiogenesis assays. We have demonstrated that within the context of the mammary gland TSP1 can modulate normal development of blood vessels. Expression of TSP1 in transgenic animals under the control of the MMTV promoter was associated with a 50-72% reduction in capillary growth. In addition, TSP1 reduced tumor size in transgenic overexpressors. The data suggest an important role for TSP1 in modulating vascular growth in both normal and pathologic tissues. The antiangiogenic region of TSP1 has been mapped to the type I (properdin) repeats. To identify novel proteins with such a domain, we have cloned two cDNAs (METH-1 and METH-2) which also have antiangiogenic properties. In addition to carboxyterminal thrombospondin-like domains they also contain metalloproteinase and disintegrin sequences. Expression of both proteins is broad but nonoverlapping. Recombinant fragments from these sequences have strong antiangiogenic potential in the CAM and cornea pocket assays. At the same molar ratio, METH-1 and METH-2 are about 20-fold more potent than TSP1. We predict that these proteins are likely endogenous modulators of vascular growth with relevant therapeutic potential in cancer and other disease states.

Endostatin is a Mr 20,000 COOH-terminal fragment of collagen XVIII that inhibits the growth of several primary tumors. We report here the cloning and expression of mouse endostatin in both prokaryotic and eukaryotic expression systems. Soluble recombinant protein expressed in yeast (15-20 mg/L) inhibited the proliferation and migration of endothelial cells in response to stimulation by basic fibroblast growth factor. A rabbit polyclonal antibody was raised that showed positive immunoreactivity to the recombinant protein expressed from both systems. Importantly, the biological activity of the mouse recombinant protein could be neutralized by this antiserum in both endothelial proliferation and chorioallantoic membrane assays. Systemic administration of endostatin at 10 mg/kg suppressed the growth of renal cell cancer in a nude mouse model. The inhibition of tumor growth with soluble yeast-produced protein was comparable to that obtained with non-refolded precipitated protein expressed from bacteria. In addition, two closely related COOH-terminal deletion mutants of endostatin were also tested and showed strikingly differing activity. Collectively, these findings demonstrate the expression of a biologically active form of mouse endostatin in yeast, define a role for the molecule in inhibiting endothelial cell migration, extend its antitumor effects to renal cell carcinoma, and provide a formal proof (via the neutralizing antiserum experiments and the mutant data) that endostatin (and not a possible contaminant) acts as an antiangiogenic agent. Finally, the high level expression of mouse endostatin in yeast serves as an endotoxin free, soluble source of protein for fundamental studies on the mechanisms of tumor growth suppression by angiogenesis inhibitors.

Formation of the blood vessel wall depends on the recruitment, proliferation, and differentiation of smooth muscle cell (SMC) precursors. The temporal events associated with the onset of expression of several SMC proteins have been well characterized in mouse and avian species. However, the timing of cell proliferation during this process has not been explored. More importantly, it has not been clear whether commitment to the smooth muscle pathway precludes proliferation during development. In the present study, we have determined the kinetics of replication in developing chick aortae between days 2.5 and 19 and have correlated these data with the expression of various SMC differentiation markers. We found that proliferation of aortic SMC precursors occurs in two waves; an early phase of rapid proliferation (15-17%; between days 4 and 12), and a second phase, when replication was reduced to less than 5% (days 16 to hatching). Proliferation of SMC during the first wave occurred concomitantly with the progressive accumulation of SMC contractile proteins, such as SM alpha-actin, calponin, myosin heavy chain, and the 1E12 antigen. We also found that the relative proliferation capacity within each compartment of the vessel wall, ie., intima, media, and adventitia varies throughout development. Approximately, 55-63% of all replicating cells were found in the tunica adventitia from days 6 to 12, whereas 35% were found in the tunica media (tunica media:adventitia = 1:2). This ratio was inverted after day 12, when most of the replicating cells were located in the tunica media (tunica media:adventitia = 2:1). In addition, we observed a ventral-to-dorsal gradient in the proliferation of SMC precursors between days 2.5 and 5. The ventral-to-dorsal proliferation gradient was similar to the previously described differential expression of two early SMC markers: alpha-actin and the 1E12 antigen. These data support the concept that a polarity exists either in the pool of SMC precursors or, in expression of factors that regulate recruitment of presumptive SMC.

Angiogenesis, the formation of new blood vessels from a pre-existing vasculature, is tightly regulated in normal adults. Under physiological circumstances, angiogenesis occurs in only a few instances; e.g., the female reproductive system in response to ovulation or gestation, the normal hair cycle, etc. In these examples, growth of new capillaries is tightly controlled by an interplay of growth regulatory proteins which act either to stimulate or to inhibit blood vessel growth. Normally, the balance between these forces is tipped in favor of inhibition and consequently capillary growth is restrained. Under certain pathological circumstances, however, local inhibitory controls are unable to restrain the increased activity of angiogenic inducers. Thus, in wound healing, inflammation and tumors, to name just a few examples, angiogenesis is integral to the pathology, engendering the hope that these pathological entities could be regulated by pharmacological and/or genetic suppression (or enhancement) of blood vessel growth. This hope, in turn, has fostered interest in the molecular mechanisms that regulate angiogenesis. In this chapter, we have reviewed the current literature regarding some angiogenic stimulators and inhibitors, emphasizing vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF), as a major angiogenic inducer, and thrombospondin (TSP) as the best known example of a natural inhibitor of vessel growth.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is an angiogenic factor with important roles in tumor growth, wound healing, and inflammation. VPF/VEGF interacts with endothelial cells by way of two high-affinity receptor tyrosine kinases: flt-1 and KDR. The vast majority of published studies have described expression of the VPF/VEGF receptors only in endothelial cells, and the statement is frequently made that these receptors are endothelial-cell-specific. In this study, we detected mRNA for flt-1 and KDR by in situ hybridization in smooth muscle cells in sections of the wall of the uterus. To confirm these unexpected findings, smooth muscle cells from the uterus and, as a control, from the colon were isolated, characterized, and cultured. Both uterine and colonic smooth muscle cells in culture expressed VPF/VEGF, but only smooth muscle cells from the uterus expressed mRNA for flt-1 and KDR by Northern analysis and in situ hybridization. Cell culture extracts of uterine but not colonic smooth muscle cells were also positive for flt-1 by Western analysis. Moreover, cultured uterine but not clonic smooth muscle cells phosphorylated the flt-1 receptor and proliferated strongly in response to added VPF/VEGF. This is one of the first rigorous demonstrations that a normal cell type other than endothelial cells can express functional receptors for VPF/VEGF in vivo and in vitro, suggesting that VPF/VEGF may have important, previously unsuspected roles on cell types other than endothelium.

The Mov-13 strain of mice was created by the insertion of the murine Moloney leukemia virus into the first intron of the alpha 1 (I) collagen gene. Consequently, Mov-13 embryos do not transcribe alpha 1 (I) collagen mRNA and lack type I collagen protein in the extracellular matrix (ECM). Homozygotes die within 12-14 days of embryonic development, in part from the rupture of large blood vessels, and also exhibit deficiencies in hematopoesis and assembly of the ECM (Lohler et al. [1984] Cell 38:597-607). Several matricellular proteins, proteoglycans, and growth factors bind to type I collagen, e.g., fibronectin, secreted protein acidic and rich in cysteine (SPARC), decorin, and transforming growth factor-beta. Here we investigate the expression and function of SPARC in the absence of type I collagen. We show that fibroblasts isolated from Mov-13 homozygous, heterozygous, and wild-type embryos transcribed and translated SPARC mRNA in vitro. However, accumulation of extracellular SPARC was severely affected in the tissues of Mov-13 homozygotes, whereas extracellular deposition of the secreted glycoproteins fibronectin and type III collagen was not altered. Since SPARC has been shown to be a regulator of cell shape, the functional consequences of the absence of extracellular SPARC were evaluated in collagen gel contraction assays. Fibroblasts isolated from homozygous Mov-13 mice did not contract native type I collagen gels as efficiently as fibroblasts from heterozygous littermates; however, addition of exogenous SPARC enhanced the contraction of collagen by homozygous Mov-13 fibroblasts. The stimulatory effect of SPARC was blocked by antibodies specific for the amino terminus of the protein. These results provide evidence that type I collagen is one of the major extracellular proteins that binds SPARC in vivo. Furthermore, the capacity of fibroblasts to contract ECM in vitro is enhanced by extracellular SPARC. We therefore propose that the remodeling of ECM by cells in vivo is regulated in part by a specific interaction between SPARC and type I collagen.

Purpose: To characterize developing retinal blood vessels with vascular markers and to relate the histochemical profile of maturing vessels to morphologic stages in retinal vascular development., Methods: Vessels were examined in frozen and paraffin-embedded retinas and in wholemounts of Macaca monkeys ranging in age from fetal day 75 (F75) to adulthood. Endothelial cells were visualized immunohistochemically using antisera to von Willebrand's factor and CD31 with lectins Ulex europaeus, Bandeiraea simplicifolia, peanut agglutinin, Ricinis communis, and wheat germ agglutinin, and by ATPase and ADPase enzymatic histochemistry. Antibodies to vascular basement membrane and matrix markers laminin, fibronectin, and collagen types I and VIII, and antisera recognizing cell cycle-specific nuclear proteins (cyclin, Ki-67, Mib-1) also were used., Results: Newly formed and mature vessels were reactive with reagents specific for CD31, von Willebrand's factor, types I and VIII collagens, laminin, fibronectin, U. europaeus, R. communis, and peanut agglutinin. Wheat germ agglutinin labeled vessels only after pretreatment with neuraminidase. All vascular markers appeared simultaneously, but some were distributed differentially between capillaries and larger vessels, along the central-peripheral extent of a vascular plexus, and among different vascular laminae. Markers of vessels failed to label spindle-shaped presumed vascular precursor cells lying peripheral to the advancing vessels during development. Spindle cells exhibited cyclin, Ki-67, and Mib-1 immunoreactivity., Conclusions: Immature and mature vitread and sclerad vessels displayed histochemical profiles that were qualitatively similar but that had subtle quantitative differences. Results do not support identification of spindle-shaped cells as vascular precursors in the developing monkey retina and are discussed in relation to mechanisms of retinal vascularization.

Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-mediated proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. This broad functional activity suggests that Vn may also play a critical role in development. To begin to investigate this possibility, we studied Vn gene expression during murine embryogenesis. In situ hybridization analysis of embryonic tissues revealed Vn mRNA primarily in the liver and the central nervous system (CNS). In the liver, Vn mRNA was detected by day 10, the level increasing at later developmental stages. In the CNS, Vn mRNA was also detected as early as day 10 and was confined to the floor plate. However, as development proceeded, high levels of Vn transcripts became prominent in the meninges of the cortex and spinal cord, and in close proximity to brain capillaries. The perikarya of most neurons lacked Vn mRNA. Unexpectedly, high levels of Vn mRNA were associated with capillaries of the CNS, but not with blood vessels of peripheral organs. These results indicate that Vn is expressed in a spatially and temporally distinct pattern during murine embryogenesis, and suggest that the Vn transcript may be a CNS-specific vascular marker.

Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

Purpose: The temporal and spatial sequence of development of laminar vascular plexuses was determined qualitatively and quantitatively in monkey and human retina., Methods: Histologic and cytochemical methods were used to study Macaca monkey eyes from fetal day 55 (F55d; birth = F168d) to 17 years, and human retina from fetal 21 weeks to adult., Results: In monkey retina, spindle-shaped, presumed vascular precursor, cells appear at F55d in the nerve fiber layer (NFL) adjacent to the optic nerve. The vascular plexuses in the NFL-ganglion cell layer appear first and form in the presence of spindle cells. Nerve fiber layer vessels extended radially to reach the temporal ora at F95d and nasal ora at F110d. The capillary plexus at the inner border of the inner nuclear layer (INL) appears at F120d near the optic disc, whereas the plexus at the outer INL border appears at F130d. Both reach their final position before birth. The INL plexuses form by endothelial budding from more vitread vessels in the absence of spindle cells. In the NFL, vessel growth to match retinal growth at the ora also involves endothelial budding. The growth rate of all plexuses was approximately 225 microns/day. The central fovea and the most peripheral retina adjacent to the ora serrata remained avascular throughout development. Differences between humans and monkeys include: Human vessels complete maturation after birth; human vessels reach the nasal ora earlier than the temporal ora; and spindle cells are more abundant and dispersed over a greater area within human NFL. Growth rates of human plexuses were comparable to those in monkeys., Conclusion: In both primates, deeper capillary plexuses form only by extension from existing vessels (angiogenesis). In the NFL, early vessel formation involves spindle precursor cells (vasculogenesis). The main difference between monkey and human in these processes is that the mature monkey vascular pattern is established well before birth.

SPARC is a secreted glycoprotein that modulates cell shape and cell-matrix interactions. Levels of SPARC are increased at sites of somitogenesis, osteogenesis, and angiogenesis in the embryo and during wound repair in the adult. We have cloned and characterized SPARC from chicken embryo. A 2.2-kbp cDNA, obtained by a novel use of the polymerase chain reaction, was determined to encode a 298-residue protein that is 85% identical to human SPARC. Antigenic sites in particular appear to be highly conserved, as antibodies against C-terminal sequences of murine and bovine SPARC reacted with a 41-43 kDa protein in chicken embryo extracts. Chicken SPARC can be defined by four sequence signatures: (a) a conserved spacing of 11 cysteine residues in domain II, (b) the pentapeptide KKGHK in domain II, which is contained within a larger region of 31 identical residues, (c) a 100% conserved region of 10 residues in domain III, and (d) a C-terminal, calcium-binding EF-hand motif. SPARC mRNAs in the 10-day-old chicken embryo are represented by three sizes of 1.8, 2.2 and 3.0 kb. The relative steady-state levels for the 2.2-kb mRNA were determined as aorta > or = skeletal muscle > calvarium > vertebra > anterior limb > kidney > heart > brain > skin and lung >> liver. The relative abundance of the 1.8-kb and 2.2-kb mRNAs varied among tissues and indicated that differential processing of SPARC mRNAs might occur. All three RNA species were detected by a cDNA probe for the N-terminal part of the coding region. Thus, the three mRNA species appear to arise from differential 3' splicing and/or polyadenylation. Collective evidence demonstrates that SPARC has been well-conserved during vertebrate evolution, a finding that indicates a fundamental role for this protein in development.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the positive regulation of angiogenesis in vivo, whereas it inhibits the proliferation of endothelial cells in vitro. To reconcile these apparently contradictory effects, we have investigated the effect of TGF-beta 1 on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro. We show that concentrations of TGF-beta 1 which stimulate proliferation of cells that form endothelial cords and/or tubes inhibit proliferation of the same cells grown at subconfluent densities. An increase in cell number of 35% over control cultures was achieved with 0.5 ng TGF-beta 1/ml. The proliferative effect was blocked by antibodies against TGF-beta. Immunological detection of BrdU-labeled nuclei revealed an increase greater than 220% in cells treated with TGF-beta 1. Moreover, a population of cells within the cords appeared to be a selective target for this cytokine. The stimulatory effect was not restricted to bovine aortic endothelial cells, as similar results were obtained with endothelial cells derived from rat microvessels. Significant levels of active TGF-beta 1 were detected in cultures containing cords/tubes, whereas only latent TGF-beta 1 was detected in subconfluent cultures. We show further that endothelial cells exhibiting angiogenesis in vitro secrete plasminogen activator, an enzyme that regulates activation of TGF-beta. The major increases in mRNA transcripts for extracellular matrix proteins that are typically associated with TGF-beta 1 were not seen in cells exhibiting angiogenesis in vitro. Since the formation of tubular networks requires both invasion and proliferation, we propose that TGF-beta 1 is a major morphoregulatory factor in angiogenesis that specifically controls endothelial cell proliferation and extracellular matrix turnover.

Thrombospondin 1 is a secreted, trimeric glycoprotein that mediates interactions between cells and extracellular matrix and exhibits cell-specific effects on migration and proliferation. Recently, two additional thrombospondin genes (thrombospondin 2 and 3) have been identified. To study the functions of these proteins, we have used in situ hybridization and RNAse protection assays to compare the expression of the genes encoding thrombospondin 1, 2, and 3 during murine embryogenesis. Thrombospondin mRNAs were associated with ossification, neuronal organogenesis, and lung development, although transcripts were differentially expressed. Thrombospondin 1 was predominant from days 10 to 13. During this period, high but transient levels of expression were observed in the neural tube, head mesenchyme, and cardiac cushions. In contrast, a more constant level of thrombospondin 1 mRNA was apparent in resident megakaryocytes of the liver, as well as in circulating megakaryocytes; neither thrombospondin 2 nor 3 was detected in these cells. Thrombospondin 1 was also produced by cells of the developing kidney and gut. The expression of thrombospondin 2 was confined principally to organized connective tissue that included pericardium, pleura, perichondrium, periosteum, meninges, ligaments, and reticular dermis. Thrombospondin 2 was also produced by differentiating skeletal myoblasts and by cells of the kidney and gut. Moreover, high levels of expression were detected in blood vessels. Thrombospondin 3 mRNA was restricted to brain, cartilage, and lung. Although thrombospondin 1, 2, and 3 belong to a family of structurally related genes, the differences observed in the spatiotemporal distribution of the corresponding mRNAs indicate unique functions for these secreted proteins.

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.