Intracardiac expression of neutral endopeptidase.

Neutral endopeptidase (NEP), a proteolytic enzyme, is known to degrade several peptides which control cardiovascular homeostasis. This is a preliminary study of the pattern of the intracardiac regional expression of the NEP gene in the normal heart, and the age-related changes in this expression in the cardiac regions. The relative abundance of NEP mRNA was determined by RT-PCR in the right atrium (RA), right ventricle (RV), left atrium (LA), left ventricle (LV) and interventricular septum (IVS) in 2-month-old (young) and 12-month-old (advanced-age adult) Wistar Kyoto (WKY) rats. The NEP gene was expressed in all 5 cardiac regions in both age groups. In young rats, the NEP expression level was lowest in the RA; this level was significantly lower than in the septum (p < 0.05). In the advanced-age adult rats, the level was lowest in the LA; this level also was significantly lower than in the septum (p < 0.05). The level in the RA in advanced-age rats was higher than that in the young rats (p < 0.01), but the levels in other regions were not significantly different between the young rats and advanced-age adult rats. Our study showed that the NEP gene was expressed in all cardiac regions of both young rats and advanced-age adult rats. However, the regional distribution of the gene was different in each age group. The region-specific expression of the NEP gene and the age-related regional changes in the expression may be due to the structural and functional characteristics of the various regions.

Urokinase activity in corneal fibroblasts may be modulated by DNA damage and secreted proteins.

Proteases like urokinase-type plasminogen activator (uPA) play an important role in tumor invasion. Cells derived from ultraviolet radiation (UVR)-induced corneal sarcomas of Monodelphis domestica produce relatively high levels of uPA compared to the untransformed keratocytes suggesting a mechanism for their invasiveness. Because UVR is known to stimulate uPA production in many cell types, UVR exposure may further increase uPA expression in corneal tumor cells, thus enhancing their ability to infiltrate. We investigated control of basal uPA levels and the induction of uPA by UVR in transformed and untransformed corneal keratocytes from Monodelphis. These studies took advantage of the fact that Monodelphis possesses an active photolyase that can be stimulated to remove UVR-induced pyrimidine dimers by exposure to long-wavelength visible photoreactivating light (PRL). Our studies showed that significant induction of uPA occurred in response to 200 J/m2 UVR. This induction was partially blocked by treatment with PRL, indicating that DNA damage, the pyrimidine dimer in particular, played a role in uPA induction. In untransformed cultured corneal fibroblasts, the heparin-binding protein inhibitor, suramin, reduced basal uPA levels, UVR-induced uPA production and cell proliferation. Basic fibroblast growth factor, a heparin-binding growth factor known to be UVR-inducible in mesenchymal cells, stimulated uPA production and cell proliferation; however, anti-bFGF antibodies did not significantly decrease proliferation or basal uPA production. These findings suggested that basal levels of uPA secretion were modulated in response to heparin-binding growth factors and that these growth factors may also have mediated the effect of UVR on uPA levels.

Cardiac chamber-specific alterations of ANP and BNP expression with advancing age and with systemic hypertension.

The present study determined cardiac chamber-specific alterations of the expression of the atrial and brain natriuretic peptide (ANP and BNP) genes with a small increase in age beyond adulthood and with systemic hypertension of intermediate duration. The expression distributions of these genes was determined using in situ hybridization in the right and left atria (RA and LA), and the right and left ventricles (RV and LV) in Wistar Kyoto rats (WKY) and age-matched Spontaneously Hypertensive rats (SHR) at ages 6 months (adult) and 8 months (advanced-age beyond adulthood). In all rat groups, both genes were expressed (ANP > BNP) in the LA and LV, and were not expressed in the RA and RV. The genes were expressed in the LA in all rat groups; the ANP, but not the BNP, expression increased with advancing age and with superimposed hypertension. They were expressed in the LV of the advanced-age WKY, adult and advanced-age SHR, but not in the adult WKY. The ANP mRNA labeling in the LA was diffuse and interspersed with dense accumulations, whereas BNP labeling was diffuse. The labeling of both genes in the form of sparse clusters was seen in the LV of the advanced-age SHR. Our study showed that ANP and BNP expression in left heart chambers increased with a small increase in age, with hypertension of intermediate duration, and with modest left ventricular hypertrophy. The chamber-specific expression distribution could be due to special groups of cardiac cells, or to local chamber-specific factors.

Degradation of type IV collagen by matrix metalloproteinases is an important step in the epithelial-mesenchymal transformation of the endocardial cushions.

Morphogenesis of some tissues and organs in the developing embryo requires the transformation of epithelial cells into mesenchyme followed by cell motility and invasion of surrounding connective tissues. Details of the mechanisms involved in this important process are beginning to be elucidated. The epithelial-mesenchymal transformation (EMT) process involves many steps, one of which is the upregulation and activation of specific extracellular proteinases including members of the matrix metalloproteinase (MMP) family. Here we analyze the role of MMPs in the initiation of the mesenchymal cell phenotype in the developing heart, and find that they are necessary for the invasion of mesenchymal cells into the extracellular matrix of the endocardial cushion tissues. An important requirement in the formation of this mesenchyme is the turnover of type IV collagen along the basal surface of endocardial cells. In vitro experiments suggest that type IV collagen does not provide a suitable migratory substrate for endocardial cushion cells unless MMP-2 and MT-MMP are active. Relevant MMPs were found to be upregulated by factors known to be involved in the induction of the EMT such as TGFbeta3. These results provide evidence of an important role for MMPs during a specific stage of the epithelial mesenchymal transformation in the embryonic heart, and suggest that specific cell-matrix interactions which facilitate cell migration only occur when the composition of the surrounding extracellular matrix is proteolytically altered.

Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the Patch mutant mouse: regulation of MMP-2 by PDGF and effects on mesenchymal cell migration.

Platelet-derived growth factors (PDGF) regulate cell proliferation, survival, morphology, and migration, as well as deposition and turnover of the extracellular matrix. Important roles for the A form of PDGF (PDGF-A) during connective tissue morphogenesis have been highlighted by the murine Patch mutation, which includes a deletion of the alpha subunit of the PDGF receptor. Homozygous (Ph/Ph) embryos exhibit multiple connective tissue defects including cleft face (involving the first branchial arch and frontonasal processes), incomplete heart septation, and heart valve abnormalities before they die in utero. Analyses of the cell biology underlying the defects in Ph/Ph embryos have revealed a deficit in a matrix metalloproteinase (MMP-2) and one of its activators (MT-MMP) that are likely to be involved in cell migration and tissue remodeling, two processes necessary for normal cardiac and craniofacial development. Morphogenesis of these structures requires infiltration of ectomesenchymal precursors and their subsequent deposition and remodeling of extracellular matrix components. First branchial arch and heart tissue from E10.5 embryos were examined by gelatin zymography and RT-PCR in order to characterize the expression of MMPs in these tissues. Of the MMPs examined, only MMP-2 and one of its activators, MT-MMP, were expressed in the first arch and heart at this stage of development. Tissues from Ph/Ph embryos exhibited a significant decrease in both MMP-2 and MT-MMP compared to tissues from normal embryos of the same developmental stage. In order to assess whether this decrease affects the motile activity of mesenchymal cells, cell migration from Ph/Ph branchial arch explants was compared to migration from normal arch tissue and found to be significantly less. In addition, the migratory ability of branchial arch cells from normal explants could be reduced in a similar manner using a specific MMP inhibitor. Although it is still unclear whether the MMP-2 reduction is a direct result of the absence of response of Ph/Ph cells to PDGF-A treatment of normal branchial arch cells in vitro with recombinant PDGF-AA significantly upregulated MMP-2 protein. Together, these results suggest that PDGF-A regulates MMP-2 expression and activation during normal development and that faulty proteinase expression may be at least partially responsible for the developmental defects exhibited by Ph/Ph embryos.

Retinal neovascularization is suppressed with a matrix metalloproteinase inhibitor.

OBJECTIVES: To determine the role of extracellular proteinases in ischemia-induced retinal neovascularization in an animal model and to examine the effect of proteinase inhibitors on retinal neovascularization. METHODS: Retinal neovascularization was induced in newborn mice exposed to 75% oxygen for 5 days, followed by room air. Retinal extracts underwent zymographic analysis to measure the activity of urokinase and matrix metalloproteinases (MMPs). Some animals under the same conditions also received intraperitoneal injections of an MMP inhibitor. Histological analysis was done to quantitate the neovascular response in these animals. RESULTS: Levels of urokinase and MMPs (MMP-2 and MMP-9) in retinas were significantly increased in animals with induced retinal neovascularization. Neovascularization was significantly inhibited with intraperitoneal administration of an MMP inhibitor. CONCLUSION: Systemic inhibition of MMPs may have therapeutic potential in preventing retinopathy associated with retinal neovascularization. CLINICAL RELEVANCE: Because up-regulation and activation of proteinases represents a final common pathway in the process of retinal neovascularization, pharmacological intervention of this pathway may be an alternative therapeutic approach to proliferative retinopathy.

Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes.

PURPOSE: Retinal neovascularization is one of the leading causes of blindness. A crucial event in this process is the remodeling and penetration of the capillary basement membrane by migrating endothelial cells. This process requires proteolysis of basement membrane components by a variety of proteinases. The objective of the present study was to determine the expression of proteinases in human retinal tissues showing active neovascularization. METHODS: Epiretinal neovascular membranes surgically removed from patients with proliferative diabetic retinopathy were analyzed by zymography, and the types and amounts of proteinases present in the tissues were determined. Retinas from nondiabetic donor eyes served as control specimens. RESULTS: Both the high- (54 kDa) and low- (33 kDa) molecular-weight forms of urokinase were present at significantly higher levels in neovascular membranes than in normal retinas. The pro forms of the matrix metalloproteinases (MMP) MMP-2 and MMP-9 were significantly elevated in the neovascular membranes in comparison with levels in normal retinas. In addition, the active forms of these enzymes were present in the membranes, whereas there was no detectable level of the active forms in normal retinas. CONCLUSIONS: Human diabetic neovascular membranes contain high levels of urokinase and MMP. The increased activity of proteinases in the final common pathway of retinal neovascularization indicates that inhibition of these enzymes may be a useful therapeutic target as an alternative approach in the management of proliferative retinopathies.

Hepatocyte growth factor expression in the developing myocardium: evidence for a role in the regulation of the mesenchymal cell phenotype and urokinase expression.

During development of the heart, the endocardium of the developing cushion tissue transforms into mesenchymal cells that rapidly invade the underlying extracellular matrix. This invasive process, along with subsequent proliferation and differentiation of the mesenchyme, are essential for the normal formation of valvular and septal structures. Several factors have been identified that appear to initiate and/or control this transformation process, including the growth factor TGF-beta. In these studies we have investigated whether hepatocyte growth factor/scatter factor (HGF/SF) may also be involved in some aspects of this transformation process. Using an immunohistochemical approach we have detected a spatially restricted localization of HGF/SF to the myocardial cells of the cushion tissue. HGF was detected in extracts of the developing heart, and the presence of the active form correlated with the onset of the transformation process and the elevation of urokinase activity. The endocardial-derived mesenchymal cells of the cushion tissue were found to express the c-met HGF receptor. Isolated endocardial cells responded to the addition of HGF with increases in motility, proliferation, and urokinase production. The results from these studies suggest that HGF may function as a myocardial-derived mediator of the epithelial-mesenchymal transformation by inducing and/or maintaining the mesenchymal cell phenotype. The increase in urokinase expression by the cushion tissue cells, in response to HGF, may facilitate the invasive abilities of these cells and also provide a means of maintaining high levels of active HGF required for the stimulation of cell proliferation and migration.

Spatial and temporal expression of the 72-kDa type IV collagenase (MMP-2) correlates with development and differentiation of valves in the embryonic avian heart.

Extracellular proteases may play an important role in the regulation of cell migration and remodeling of the extracellular matrix during development. In this study, we have examined the embryonic avian heart for the expression of matrix metalloproteases. The 72-kDa type IV collagenase, MMP-2, was detected in extracts of whole hearts and showed a modest increase in amount over time. This increase in enzyme activity corresponded to a small increase in the steady-state level of mRNA for this enzyme. A more dramatic increase was seen in the amount of the 66-kDa activated form of this enzyme as development progressed, suggesting that the process of activation, rather than enzyme synthesis, may be the important regulatory step in this system. Coincident with the change in the level of active MMP-2 was a significant increase in the expression of the MMP-2 activator, MT-MMP, between stages 12 and 24. The message for MMP-2 was expressed by the endocardium of the cushion tissues which was undergoing an epithelial-mesenchymal transition, and by the migrating mesenchymal cells, suggesting a role for this protease in regulating cell motility and matrix invasion. In older staged hearts, the cells of the differentiating valves expressed high levels of MMP-2 which may be important for the final remodeling events in this region.

Regulation of urokinase expression in the developing avian heart: a role for the Ets-2 transcription factor.

During heart development, cells of the endocardial cushions undergo an epithelial-mesenchymal transformation and migrate into the surrounding extracellular matrix. This event is required for the normal formation of valves and chamber septation. Coincident with this phenotypic change is the expression of the serine protease urokinase by the mesenchymal cells. This protease plays an important role in remodeling of the matrix, promotion of cell migration by regulating cell-matrix interactions, and the activation of growth factors. To understand the mechanisms underlying the expression of urokinase during heart development, studies were designed to analyze the role of the Ets transcription factors in the regulation of the avian urokinase gene promoter. Deletion or mutagenesis of the Ets consensus sites significantly decreased the activity of the promoter in isolated cushion tissue cells. Proteins were identified by electrophoretic mobility shift analysis and UV-crosslinking which bound to a specific region of the promoter shown to be required for full transcriptional activity. Analyses based upon protein molecular weight and interaction with specific antibodies suggest a role for the Ets-2 protein in promoter binding and activity. The expression of Ets-2 in the cushion tissue cells was confirmed by RT-PCR analysis and in situ hybridization. The mRNA levels and the DNA binding activity of the Ets-2 protein were found to change during development paralleling the increase in urokinase activity. Overexpression of the full-length Ets-2 protein or a dominant-negative form of the protein altered the activity of the promoter and significantly affected the production of urokinase in these cells. The results from these studies suggest an important role for the Ets-2 protein in heart development and may contribute to a better understanding of the inductive factors present in the heart which facilitate the normal morphogenesis of this organ.

Composition in situ and in vitro of vascular smooth muscle laminin in the rat.

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.

Injury-induced 92-kilodalton gelatinase and urokinase expression in rat brain.

BACKGROUND: Proteolytic disruption of the extracellular matrix is important in pathologic processes. We have shown that activated 72-kilodalton (kd) type IV collagenase injected intracerebrally attacks brain extracellular matrix and opens the blood-brain barrier. Therefore, we tested the hypothesis that endogenous production of matrix-degrading proteases may be a factor in secondary brain injury. EXPERIMENTAL DESIGN: Adult rats had a hemorrhagic injury produced by injection of 0.4 units of bacterial collagenase into the caudate/putamen. Endogenous production of matrix metalloproteinases and plasminogen activators (PA) was measured by substrate-gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (zymography) at 1, 4, 8, 16, 24, and 48 hours, and 7 and 14 days after the injury. RESULTS: Gelatin-containing zymograms had bands of the expected molecular weights from the injected bacterial collagenase at 1 hour. By 8 hours a new 92-kd gelatinase was seen in zymograms. EDTA eliminated the 92-kd band, indicating that it was a metalloproteinase. The 92-kd type IV collagenase/gelatinase was maximally increased by 24 hours (p < 0.0001). Plasminogen-casein zymography showed 40- and 60-kd bands from PA. The 40-kd PA reached a maximum at 24 hours (p < 0.05) and remained elevated for 7 days. Amiloride completely eliminated the 40-kd band and reduced the 60-kd band, suggesting that they were a urokinase-type PA. CONCLUSIONS: Hemorrhagic injury induces both 92-kd type IV collagenase/gelatinase and 40-kd urokinase-plasminogen activator expression in brain. We propose that metalloproteinases and serine proteases take part in a proteolytic cascade that breaks down extracellular matrix, opening the blood-brain barrier with secondary brain edema and cell death.

Transforming growth factor-beta 1 differentially regulates proliferation, morphology, and extracellular matrix expression by three neural crest-derived neuroblastoma cell lines.

We reported previously (S. L. Rogers, P. J. Gegick, S. M. Alexander, and P. G. McGuire, Dev. Biol. 151, 191-203, 1992) that transforming growth factor-beta 1 (TGF beta 1) inhibited proliferation, up-regulated fibronectin synthesis, and suppressed melanogenesis in a population of quail neural crest cells in vitro. Here, we report that cell lines derived from the parent SK-N-SH neuroblastoma line (R. A. Ross, B. A. Spengler, and J. L. Biedler, J. Natl. Cancer Inst. 71, 741-747, 1983) respond differentially to TGF beta 1, and their responses provide further insights into the actions of this growth factor on neural crest subpopulations. The SH-EP cell line exhibits primarily nonneuronal traits and responded to TGF beta 1 with increased thymidine uptake after 6 days of culture, increased expression of fibronectin mRNA and protein, and decreased laminin synthesis. Many SH-EP cells also acquired a dramatically elongated morphology, reminiscent of Schwann cells in culture. Thymidine uptake by the neuronal SY5Y cell line was not substantially altered. Neither fibronectin mRNA nor protein was detectable in either TGF beta 1-treated or untreated cultures, although laminin synthesis was upregulated by the growth factor. In TGF beta 1-treated cultures of the intermediate SH-IN cell line, which has been reported to display both neuronal and nonneuronal characteristics, there was marked flattening of many cells, a steady decrease in thymidine uptake, and increased expression of both fibronectin and laminin. The observed responses of SH-IN cells mimic those observed in primary neural crest cultures and appear to represent similar differentiation toward a mesenchymal phenotype. These results substantiate the idea that closely related but diverging neural crest-derived cell types respond selectively to TGF beta 1 and demonstrate that these SK-N-SH-derived cell lines will be useful in experimental approaches that will allow us to infer mechanisms underlying regulation of neural crest differentiation.

Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi.

The endogenous biosynthesis of nitric oxide (NO) is increased during gestation. To begin our investigation of a possible tissue source (or sources), we examined the placenta. We postulated that analogous to the endothelium of blood vessels, the syncytiotrophoblast (STr) cell layer that lines the intervillous blood space of the human placenta would express NO synthase. Our results show that human placental villi express a calcium- and calmodulin-sensitive form of NO synthase, located mainly in the microsomal cell fraction. By in situ hybridization using a riboprobe generated from human endothelial NO synthase cDNA, we observe NO synthase mRNA expression in STr. The STr also shows NADPH-diaphorase staining, indicating the presence of NO synthase, and most likely other flavin-containing enzymes involved in sex steroid metabolism. NO synthase activity was also detected in the villi of a complete mole placenta (which lacks fetal vessels), further supporting a trophoblastic origin. Our findings suggest a previously unrecognized role for STr-derived NO in placental function.

Inhibition of urokinase synthesis and cell surface binding alters the motile behavior of embryonic endocardial-derived mesenchymal cells in vitro.

The expression of the serine protease urokinase is elevated during the epithelial-mesenchymal transformation of the endocardium in the developing avian heart. Elevated urokinase expression is associated with the migrating mesenchymal cells of the atrioventricular canal and bulbotruncus and not the myocardium. Treatment of isolated endocardial-derived mesenchymal cells with phosphatidyinositol-specific phospholipase C released urokinase and its receptor from the cell surface and caused significant alterations in cell morphology and motility. Likewise inhibition of urokinase synthesis by treatment of cells with antisense oligonucleotides also inhibited the migration and motility of the endocardial-derived cells. These results suggest an important role for this enzyme in cell-matrix interactions and cell migration during development.

Urokinase production by embryonic endocardial-derived cells: regulation by substrate composition.

Early events in cardiovascular morphogenesis are characterized by cell migrations and extensive tissue remodeling. We are interested in the role played by the extracellular serine protease urokinase in these events. Elevated urokinase activity and mRNA levels have been shown to be associated with the onset of ventricular trabeculation and mesenchymal cell migration in the endocardial cushion tissues of the atrioventricular canal and the outflow tract of the quail embryo. In this study, urokinase production by isolated endocardial-derived cells was found to be affected by the composition of the matrix to which the cells were exposed. Interaction of cells with a 45-kDa gelatin-binding fragment of fibronectin upregulated the production of urokinase by nearly threefold. This increase in urokinase activity had profound influences on cell motility and spreading.

Isolation and culture of rat superior mesenteric artery smooth muscle cells.

The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.

Urokinase expression during the epithelial-mesenchymal transformation of the avian somite.

Early events in the morphogenesis of the axial skeleton involve an epithelial-mesenchymal transformation of the somites. Cells of the ventromedial wall of the somite (the sclerotome) migrate to regions surrounding the notochord and neural tube and condense to form the cartilage model of the vertebrae. Urokinase activity in the axial region of the quail embryo trunk was found to increase during these stages. In situ hybridization localized urokinase mRNA expression in this region and suggests an important role for this protease in the process of cell migration and matrix remodeling during development of the axial skeleton.

Transforming growth factor-beta alters differentiation in cultures of avian neural crest-derived cells: effects on cell morphology, proliferation, fibronectin expression, and melanogenesis.

Neural crest cell differentiation is responsive to a variety of extrinsic signals that include extracellular matrix (ECM) molecules and growth factors. Transforming growth factor-beta (TGF-beta) has diverse, cell type-specific effects, many of which involve regulation of synthesis of ECM molecules and their cell surface receptors. We are studying both separate and potentially interrelated influences of ECM and growth factors on crest differentiation and report here that TGF-beta alters several aspects of crest cell behavior in vitro. Clusters of quail neural crest cells were cultured in the presence and absence of 400 pM TGF-beta 1 and examined at 1, 3, and 5 days. When examined at 5 days, there was a dramatic decrease in the number of melanocytes in treated cultures, regardless of the onset or duration of TGF-beta treatment. With continuous TGF-beta treatment, or with treatment only during crest cluster formation on explanted neural tubes, many cells increased in area, becoming extremely flat. These changes were evident beginning on Day 3. While quantitative analyses of video images documented the size increase, several aspects of motility were relatively unchanged. Synthesis of fibronectin (FN) by approximately 11% of cells on Day 3 and 31% of cells on Day 5 was demonstrated by immunocytochemistry and was associated with a sixfold increase in FN mRNA by Day 5. Experiments which correlated FN immunoreactivity with incorporation of bromodeoxyuridine suggested that the population of large, flat, FN-positive cells did not proliferate selectively and that there was a slower rate of proliferation in TGF-beta-treated cultures than in untreated cultures. The large FN-immunoreactive cells resemble cells derived from cephalic neural crest and raise interesting questions concerning potential roles for TGF-beta in regulating crest differentiation in vivo.

Urokinase activity in the developing avian heart: a spatial and temporal analysis.

Early events in cardiac morphogenesis are characterized by cell migrations and extensive tissue remodeling. This study was undertaken to determine the levels of urokinase in specific regions of the avian heart during early stages of development. Urokinase has previously been shown to be involved in both cell migration and matrix turnover. Elevated urokinase activity and mRNA levels were associated with the onset of ventricular trabeculation and mesenchymal cell migration in the endocardial cushion tissues. Urokinase was localized by immunostaining to the endocardial and mesenchymal cells of the developing atrioventricular canal (AVC) and outflow tract (OFT) as well as with evaginating ventricular endocardium. No immunoreactivity was seen associated directly with the matrix, suggesting that the enzyme remained mostly cell associated, a finding which was confirmed in isolated endocardial cells. Results from this study suggest a role for urokinase in the tissue remodeling and cell migration that occurs during the early stages of cardiac morphogenesis.