Vasomotor control in arterioles of the mouse cremaster muscle.

Recent advances in transgenic mouse technology provide novel models to study cardiovascular physiology and pathophysiology. In light of these developments, there is an increasing need for understanding cardiovascular function and blood flow control in normal mice. To this end we have used intravital microscopy to investigate vasomotor control in arterioles of the superfused cremaster muscle preparation of anesthetized C57Bl6 mice. Spontaneous resting tone increased with branch order and was enhanced by oxygen. Norepinephrine and acetylcholine (ACh) caused concentration-dependent vasoconstriction and vasodilation, respectively. Microiontophoresis of ACh evoked vasodilation that conducted along arterioles; the local (direct) response was inhibited by N(omega)-nitro-L-arginine (LNA), and both local and conducted responses were inhibited by 17-octadecynoic acid (17-ODYA). Microejection of KCl evoked a biphasic response: a transient conducted vasoconstriction (inhibited by nifedipine), followed by a conducted vasodilation that was insensitive to LNA, indomethacin, and 17-ODYA. Phenylephrine evoked focal vasoconstriction that did not conduct. Perivascular sympathetic nerve stimulation evoked constriction along arterioles that was inhibited by tetrodotoxin. These findings indicate that for arterioles in the mouse cremaster muscle, nitric oxide and endothelial-derived hyperpolarizing factor (as shown by LNA and 17-ODYA interventions, respectively) mediate vasodilatory responses to ACh but not to KCl, and that vasomotor responses spread along arterioles by multiple pathways of cell-to-cell communication.

Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall.

The assembly of the vessel wall from its cellular and extracellular matrix components is a critical process in the development and maturation of the cardiovascular system. However, fundamental questions concerning the origin of vessel wall cells and the mechanisms that regulate their development and differentiation remain unanswered. The initial step of vessel wall morphogenesis is formation of a primary vascular network, comprised of nascent endothelial cell tubes, via the processes of vasculogenesis and angiogenesis. Subsequently, primordial vascular smooth muscle cells (VSMCs) are recruited to the endothelium to form a multilayered vessel wall. During the course of development and maturation, the VSMC plays diverse roles: it is a biosynthetic, proliferative, and contractile component of the vessel wall. Although the field of vascular development has blossomed in the past decade, the molecules and mechanisms that regulate this developmental pathway are not well defined. The focus of this review is on those facets of VSMC development important for transforming a nascent endothelial tube into a multilayered structure. We discuss the primordial VSMC with particular attention to its purported origins, the components of the extracellular milieu that contribute to its development, and the contribution of embryonic hemodynamics to vessel wall assembly.

Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence.

The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.

Morphogenesis of the first blood vessels.

The initial phase of vessel formation is the establishment of nascent endothelial tubes from mesodermal precursor cells. Development of the vascular epithelium is examined using the transcription factor TAL1 as a marker of endothelial precursor cells (angioblasts), and a functional assay based on intact, whole-mounted quail embryos. Experimental studies examining the role(s) of integrins and vascular endothelial growth factor (VEGF) establish that integrin-mediated cell adhesion is necessary for normal endothelial tube formation and that stimulation of embryonic endothelial cells with exogenous VEGF results in a massive "fusion" of vessels and the obliteration of normally avascular zones. The second phase of vessel morphogenesis is assembly of the vessel wall. To understand the process by which mesenchyme gives rise to vascular smooth muscle, a novel monoclonal antibody, 1E12, that recognizes smooth muscle precursor cells was used. Additionally, development of the vessel wall was examined using the expression fo extracellular matrix proteins as markers. Comparison of labeling patterns of 1E12 and the extracellular matrix molecules fibulin-1 and fibrillin-2 indicate vessel wall heterogeneity at the earliest stages of development; thus smooth muscle cell diversity is manifested during the differentiation and assembly of the vessel wall. From these studies it is postulated that the extracellular matrix composition of the vessel wall may prove to be the best marker of smooth muscle diversity. The data are discussed in the context of recent work by others, especially provocative new studies suggesting an endothelial origin for vascular smooth muscle cells. Also discussed is recent work that provides clues to the mechanism of vascular smooth muscle induction and recruitment. Based on these findings, vascular smooth muscle cells can be thought of as existing along a continuum of phenotypes. This spectrum varies from mainly matrix-producing cells to primarily contractile cells; thus no one cell type typifies vascular smooth muscle. This view of the smooth muscle cell is considered in terms of a contrasting opinion that views smooth muscle cell as existing in either a synthetic or proliferative state.

Proliferation and differentiation of smooth muscle cell precursors occurs simultaneously during the development of the vessel wall.

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.

Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

The development of the coronary vessels and their differentiation into arteries and veins in the embryonic quail heart.

Research concerning the embryologic development of the coronary plexus has enriched our understanding of anomalous coronary vessel patterning. However, the differentiation of the coronary vessel plexus into arteries, veins, and a capillary network is still incomplete. Immunohistochemical techniques have been used for whole mounts and serial sections of quail embryo hearts to demonstrate endothelium, vascular smooth muscle cells, and fibroblasts. From HH35 onward, the lumen of the coronary plexus was visualized by injecting India ink into the aorta. In HH17, branches from the sinus venosus plexus expand into the proepicardial organ to reach the dorsal side of the atrioventricular sulcus. From HH25 onward, vessel formation proceeds toward the ventral side and the apex of the heart. After lumenized connections of the coronary vessels with the aorta and right atrium are established, a media composed of smooth muscle cells and an adventitia composed of procollagen-producing fibroblasts are formed around the coronary arteries. In the early stage, bloodflow through the coronary plexus is possible, although connections with the aorta have yet to be established. After the coronary plexus and the aorta and right atrium are interconnected, coronary vessel differentiation proceeds by media and adventitia formation around the proximal coronary arteries. At the same time, the remodeling of the vascular plexus is manifested by disappearance of arteriovenous anastomoses, leaving only capillaries to connect the arterial and venous system.

Differences in development of coronary arteries and veins.

OBJECTIVE: The differentiation of the coronary vasculature was studied to establish in particular the formation of the coronary venous system. METHODS: Antibody markers were used to demonstrate endothelial, smooth muscle, and fibroblastic cells in serial sections of embryonic quail hearts. The anti-beta myosin heavy chain and the neuronal marker HNK-1 were added to our incubation protocol. RESULTS: In HH32, the coronary vascular network has developed into a circulatory system with connections to the sinus venosus, the aorta and the right atrium. The connections between the aorta and the right atrium allow for direct arteriovenous shunting. Subsequently, differentiation into coronary arteries and veins occurs with an interposed capillary network. The smooth muscle cells of the coronary arterial media derive from the subepicardial layer, whereas the subepicardially located cardiac veins recrute atrial myocardium, as these cells express the beta-myosin heavy chain antigen. Ganglia are located in the subepicardium close to the vessels, while nerve fibres tend to colocalize with the formed vessel channels. CONCLUSIONS: A new finding is presented in which the subepicardial coronary veins have a media that consists of myocardial cells. The close positional relationship of neural tissue and coronary vessels that penetrate the heart wall is explained as inductive for vessel wall differentiation, but not for invasion into the heart.

Inhibition of pp125FAK in cultured fibroblasts results in apoptosis.

The tyrosine kinase called pp125FAK is believed to play an important role in integrin-mediated signal transduction. pp125FAK is associated both functionally and spatially with integrins, which are the cell surface receptors for extracellular matrix components. Although the precise function of pp125FAK is not known, two possibilities have been proposed: pp125FAK may regulate the assembly of focal adhesions in spreading or migrating cells, or pp125FAK may participate in a signal transduction cascade to inform the nucleus that the cell is anchored. To test these models in living cells, a peptide representing the focal adhesion kinase (FAK)-binding site of the beta 1 tail was coupled to carrier protein and injected into cultured cells to competitively inhibit the binding of pp125FAK to endogenous integrin, thus inhibiting activation of pp125FAK on a cell-by-cell basis. In addition, an antibody directed against an epitope adjacent to the focal adhesion targeting sequence on pp125FAK was microinjected, as an alternative means of inhibiting pp125FAK activation. It was observed that when rounded cells were injected with either the integrin peptide or the anti-FAK antibody, the cells rapidly began to apoptose, within 4 h after injection. These results indicate that pp125FAK may play a critical role in suppressing apoptosis in fibroblasts.

Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers.

The building of the vessel wall from its cellular and extracellular matrix (ECM) components is a critical event in the development and maturation of the cardiovascular system. However, little is known about the events that occur after the initial vascular network, a nascent endothelium, is established. The proper recruitment of vascular smooth muscle cells (VSMCs) to the endothelium is one such critical event. Although the majority of VSMCs are of mesodermal origin, it is not understood which populations of embryonic cells are capable of following the VSMC differentiation pathway. Previous studies, which have focused on the VSMC component of vessel wall development, have been limited by the use of markers that are not smooth muscle specific, or have focused on events that occur after a multilayered wall has been established. Therefore, the initial goal of this study was to define when overtly identifiable VSMCs were first associated with the vascular endothelium. Monoclonal antibodies (MAbs) were generated from embryonic vessel wall antigens in order to circumvent problems of cell specificity associated with the use of previously available markers to VSMCs. Critical to this study is our MAb, 1E12, which unlike other antibody markers, is smooth muscle specific. Using 1E12, we defined a pattern for recruitment and differentiation of the VSMC component of the descending aorta in stage 12 to stage 20 (Hamburger and Hamilton, 1951) quail embryos. Immunofluorescent labeling of quail embryos with 1E12 and a MAb to smooth muscle alpha-actin (SM alpha A) shows that the first mesodermally derived cells to associate with the aortic endothelium do so at the ventral surface. Recruitment of these cells, which we believe to be primordial VSMCs, proceeds in a ventral to dorsal direction along the aorta and in a radial direction, emanating from the endothelium. Additionally, we have determined the distribution of several ECM proteins, during the initial events of vessel wall development. Our studies show that fibulin-1 is expressed surrounding the primordial VSMCs of the vessel wall before elastin precursors are present and suggest that differential expression of the JB3 antigen (Wunsch et al., 1994) may be indicative of early diversity among embryonic VSMCs.

Fibulin is localized at sites of epithelial-mesenchymal transitions in the early avian embryo.

Fibulin is a 100-kDa calcium-binding, extracellular matrix (ECM), and plasma glycoprotein (Argraves et al., Cell 58, pp. 623-629, 1989; Argraves et al., J. Cell Biol. 111, 3155-3164). Immunoprecipitation analysis showed that antibodies against human fibulin react with an avian isoform (M(r) 100,000). The spatial and temporal distribution of fibulin was examined in the early avian embryo using immunofluorescence microscopy. In stage 15-22 quail embryos fibulin is a constituent of most basement membranes. Areas undergoing epithelial-mesenchymal transitions such as the endocardial cushions, developing myotomes, and neural crest display especially prominent immunostaining. In the early heart fibulin expression was most pronounced in the cardiac jelly at sites where endocardial cushion cells begin the migrations that lead to the formation of valvular and septal primordia. Laser scanning confocal microscopy showed extensive extracellular accumulations of fibulin on the surface of endocardial mesenchyme cells that were motile at the time of fixation (stage 19). These data suggest that enhanced deposition of fibulin at sites of epithelial-mesenchymal transitions may influence cell behavior.

Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.