Dynamic physiological temperature and pressure sensing with phase-resolved low-coherence interferometry.

We report the development and characterisation of highly miniaturised fibre-optic sensors for simultaneous pressure and temperature measurement, and a compact interrogation system with a high sampling rate. The sensors, which have a maximum diameter of 250 µm, are based on multiple low-finesse optical cavities formed from polydimethylsiloxane (PDMS), positioned at the distal ends of optical fibres, and interrogated using phase-resolved low-coherence interferometry. At acquisition rates of 250 Hz, temperature and pressure changes of 0.0021 °C and 0.22 mmHg are detectable. An in vivo experiment demonstrated that the sensors had sufficient speed and sensitivity for monitoring dynamic physiological pressure waveforms. These sensors are ideally suited to various applications in minimally invasive surgery, where diminutive lateral dimensions, high sensitivity and low manufacturing complexities are particularly valuable.

Extracellular matrix fluctuations during early embryogenesis.

Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node).

Multi-field 3D scanning light microscopy of early embryogenesis.

A computer-controlled microscopy system was devised to allow the observation of avian embryo development over an extended time period. Parallel experiments, as well as extended specimen volumes, can be recorded at cellular resolution using a three-dimensional scanning procedure. The resulting large set of data is processed automatically into registered, focal- and positional-drift corrected mosaic images, assembled as montages of adjacent microscopic fields. The configuration of the incubator and a sterile embryo chamber prevents condensation of the humidified culturing atmosphere in the optical path and is compatible with both differential interference contrast and epifluorescence optics. As a demonstration, recordings are presented showing the large-scale remodelling of the embryonic primordial vascular structure.

Integrins in vascular development.

In recent years, there has been a sustained interest in vascularization processes. Much, if not all, of the work has included the concept of new vessel morphogenesis. Surprisingly, most of the work has not addressed developmental mechanisms directly, but rather as an offshoot of a disease process, wound healing process, or from the perspective of inducing vessels in an ischemic site. One theme has dominated the various studies on capillary or endothelial tube morphogenesis-integrin-mediated cell behavior. Integrin biology impacts virtually every known step of nascent vessel formation. In this review article, we attempted to summarize key findings from the viewpoint of developmental biologists/morphologists. We also attempted to summarize and contrast data obtained using integrin gene ablation approaches in mice with other experimental systems. It is hoped this review will provide a distinct cell biological perspective to vascular scientists from the clinical, molecular, and tissue engineering communities.

VEGF regulates cell behavior during vasculogenesis.

Prominent among molecules that control neovascular processes is vascular endothelial growth factor (VEGF). The VEGF ligands comprise a family of well-studied mitogens/permeability factors that bind cell surface receptor tyrosine kinases. Targets include VEGF receptor-1/Flt1 and VEGF receptor-2/Flk1. Mice lacking genes for VEGF ligand or VEGF receptor-2 die early in gestation, making it difficult to determine the precise nature of underlying endothelial cellular behavior(s). To examine the effect(s) of VEGF signaling on cell behavior in detail, we conducted loss-of-function studies using avian embryos. Injection of soluble VEGFR-1 results in malformed vascular networks and the absence of large vessels. In the most severe cases embryos exhibited vascular atresia. Closely associated with the altered phenotype was a clear endothelial cell response-a marked decrease in cell protrusive activity. Further, we demonstrate that VEGF gain of function strikingly increased cell protrusive activity. Together, our data show that VEGF/VEGF receptor signaling regulates endothelial cell protrusive activity, a key determinant of blood vessel morphogenesis. We propose that VEGF functions as an instructive molecule during de novo blood vessel morphogenesis.

VEGF and vascular fusion: implications for normal and pathological vessels.

The avian embryo is well suited for the study of blood vessel morphogenesis. This is especially true of investigations that focus on the de novo formation of blood vessels from mesoderm, a process referred to as vasculogenesis. To examine the cellular and molecular mechanisms regulating vasculogenesis, we developed a bioassay that employs intact avian embryos. Among the many bioactive molecules we have examined, vascular epithelial growth factor (VEGF) stands out for its ability to affect vasculogenesis. Using the whole-embryo assay, we discovered that VEGF induces a vascular malformation we refer to as hyperfusion. Our studies showed that microinjection of recombinant VEGF165 converted the normally discrete network of embryonic blood vessels into enlarged endothelial sinuses. Depending on the amount of VEGF injected and the time of postinjection incubation, the misbehavior of the primordial endothelial cells can become so exaggerated that for all practical purposes the embryo contains a single enormous vascular sinus; all normal vessels are subsumed into a composite vascular structure. This morphology is reminiscent of the abnormal vascular sinuses characteristic of certain neovascular pathologies. (J Histochem Cytochem 47:1351-1355, 1999)

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.

Identification of the developmental marker, JB3-antigen, as fibrillin-2 and its de novo organization into embryonic microfibrous arrays.

The monoclonal antibody JB3 was previously shown to react with a protein antigen present in the bilateral primitive heart-forming regions and septation-stage embryonic hearts; in addition, primary axial structures at primitive streak stages are JB3-immunopositive (Wunsch et al. [1994] Dev. Biol. 165:585-601). The JB3 antigen has an overlapping distribution pattern with fibrillin-1, and a similar molecular mass (Gallagher et al. [1993] Dev. Dyn. 196:70-78; Wunsch et al. [1994] Dev. Biol. 165:585-601). Here we present immunoblot and immunoprecipitation data showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harvested using JB3-immunoaffinity chromatography. A single polypeptide (Mr = 350,000), which was not immunoreactive with an antibody to fibrillin-1, eluted from the affinity column. Mass spectroscopy peptide microsequencing determined the identity of the JB3 antigen to be an avian homologue of fibrillin-2. Live, whole-mounted, quail embryos were immunolabeled using a novel microinjection approach, and subsequently fixed. Laser scanning confocal microscopy indicated an elaborate scaffold of fibrillin-2 filaments encasing formed somites. At more caudal axial positions, discrete, punctate foci of immunofluorescent fibrillin-2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully-formed somites, progressively longer filamentous assemblies of fibrillin-2 were observed, suggesting a developmental progression of fibrillin-2 fibril assembly across the somite-forming region of avian embryos. Extensive filaments of fibrillin-2 connect somites to the notochord. Similarly, fibrillin-2 connects the mesoderm associated with the anterior intestinal portal to the midline. Thus, fibrillin-2 fibrils are organized by a diverse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin-2 microfilaments are assembled in a temporal and spatial pattern that is coincident with cranial-to-caudal segmentation, and regression of the anterior intestinal portal. Fibrillin-2 may function to impart physical stability to embryonic tissues during morphogenesis of the basic vertebrate body plan.

Identification of chicken and C. elegans fibulin-1 homologs and characterization of the C. elegans fibulin-1 gene.

Fibulin-1, a member of the emerging family of fibulin proteins, is a component of elastic extracellular matrix fibers, basement membranes and blood. Homologs of fibulin-1 have been described in man, mouse and zebrafish. In this study, we describe the isolation and sequencing of chicken fibulin-1C and D cDNA variants. We also describe identification of a C. elegans cDNA encoding fibulin-1D and cosmids containing the C. elegans fibulin-1 gene. Using the cDNA, RT-PCR and computer-based analysis of genomic sequences, the exon/intron organization of the C. elegans fibulin-1 gene was determined. The C. elegans fibulin-1 gene is located on chromosome IV, is approximately 6 kb in length, contains 16 exons and encodes fibulin-1C and D variants. Comparative analysis of the deduced amino acid sequences of nematode and chicken fibulin-1 variants with other known vertebrate fibulin-1 polypeptides showed that the number and organization of structural modules are identical. The results of this study indicate that the structure of the fibulin-1 protein has remained highly conserved over a large period of evolution, suggestive of functional conservation.

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.

TAL1/SCL is expressed in endothelial progenitor cells/angioblasts and defines a dorsal-to-ventral gradient of vasculogenesis.

In this study we establish that TAL1/SCL, a member of the helix-loop-helix family of transcription factors, and an important regulator of the hematopoietic lineage in mice, is expressed in the endothelial lineage of avians. The earliest events of vascular development were examined using antibodies to TAL1/SCL, and the QH1 antibody, an established marker of quail endothelial cells. Analyses using double immunofluorescence confocal microscopy show that: (i) TAL1/SCL is expressed by both quail and chicken endothelial cells; (ii) TAL1/SCL expression precedes that of the QH1 epitope; and (iii) TAL1/SCL, but not QH1, expression defines a subpopulation of primordial cells within the splanchnic mesoderm. Collectively these data suggest that TAL1/SCL-positive/QH1-negative cells are angioblasts. Further, using TAL1/SCL expression as a marker of the endothelial lineage, we demonstrate that in addition to the previously described cranial-to-caudal gradient, there is a dorsal-to-ventral progression of vasculogenesis.

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.

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-1, vitronectin, and fibronectin expression during avian cardiac valve and septa development.

BACKGROUND: Extracellular matrix (ECM) proteins have been implicated as mediators of events important to valvuloseptal development (reviewed by Little and Rongish, Experentia, 51:873-882, 1995). The aim of this study was to identify connective tissue ECM proteins present at sites of valvuloseptal morphogenesis, and to determine how their patterns of expression change during the developmental process. METHODS: Immunofluorescence microscopy was used to examine the distribution of fibulin-1, vitronectin, and fibronectin in the embryonic chicken heart over a broad developmental time frame (Hamburger and Hamilton stages 14 to 44), emphasizing stages that illustrate endocardial cushion formation, growth, fusion, and development into valvuloseptal components. RESULTS AND CONCLUSIONS: Fibulin-1 immunolabeling was concentrated in endocardial cushions, notably at boundaries with the myocardium, during stages when the cushions are differentiating into valvular and septal components. Fibulin-1 was detected in the endocardial cushions prior to their seeding with cushion cells, but became undetectable by early midgestation. Vitronectin expression was similar to fibulin-1, but less restricted in its distribution. Vitronectin was observed before endocardial cushion cell migration commenced and persisted until the formation of prevalvular structures (early midgestation) in the atrioventricular cushions. Vitronectin remained detectable in the semilunar valves until late midgestation. Fibronectin was present in the endocardial cushion region and in portions of the endocardium and myocardium throughout the stages presented. Our data suggests that the ECM of the endocardial cushions undergoes remodelling in a regionally and temporally specific manner which corresponds with morphogenetic changes during valvuloseptal development.

Distribution of connective tissue proteins during development and neovascularization of the epicardium.

OBJECTIVE: The epicardium is the site of initial cardiac neovascularization and formation of the coronary circulatory system. Recent evidence indicates that vascular progenitor cells are influenced by the connective tissue proteins of their extracellular environment, yet little is known about the composition or function of the embryonic epicardial extracellular matrix (ECM). This study examines the distribution of ECM proteins during the migration, growth and maturation of epicardial cells and also during the development of the coronary vascular network. METHODS: Immunofluorescence microscopy was used to determine the distributions of vitronectin, fibronectin and a newly described fibrillin-like protein, the JB3 antigen, in the embryonic chicken heart. Immunoblot analysis was performed to compare the relative electrophoretic mobilities of the JB3 antigen and fibrillin-1. RESULTS: The data show that vitronectin and fibronectin are present at sites of initial migration of the epicardial cells. The expression of vitronectin (and also fibronectin) becomes more pronounced as the epicardium thickens, undergoes remodeling and differentiates. The JB3 antigen is prominently expressed in the coronary arteries, allowing visualization of their connection to the systemic circulation and to the heart muscle, as well as vessel wall formation and organization. Immunoblot analysis suggests that the JB3 antibody recognizes a fibrillin-like polypeptide that is distinct from fibrillin-1. CONCLUSIONS: The observed distributions of vitronectin and fibronectin are consistent with roles in migration of epicardial cells, in remodeling of the epicardium and as substratum components during blood vessel formation. The observed distribution of the JB3 antigen indicates a structural/organizational role in coronary arterial wall assembly and suggests that the JB3 antibody be considered an early marker for maturing coronary arteries.

Use of Starvation Promoters To Limit Growth and Selectively Enrich Expression of Trichloroethylene- and Phenol-Transforming Activity in Recombinant Escherichia coli.

Volume 61, no. 9, p. 3323: the title of the article should read as shown above. [This corrects the article on p. 3323 in vol. 61.].