Fibroblast growth factor 2 control of vascular tone.

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FGF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

The effects of endocrine and mechanical stimulation on stage I lactogenesis in bovine mammary epithelial cells.

The study objective was to evaluate the effect of endocrine and mechanical (gel release) signaling on bovine mammary epithelial cell ultrastructure and gene expression. Cultures receiving only one stimulus demonstrated partially differentiated ultrastructure, which included abundant polysomes, limited rough endoplasmic reticulum, and absence of secretory products, whereas the 2 stimuli together induced a more complete lactogenic phenotype that included increased rough endoplasmic reticulum, abundant lipid droplets, and secretory vesicles containing casein micelles. The structural data indicated that although synthesis of milk components was initiated, the copious synthesis and secretion associated with stage II lactogenesis was not evident. Microarray analysis revealed that both prolactin and gel release independently regulated several genes linked to a wide array of cellular activities. In combination, they regulated fewer genes targeted to lactogenesis. Genes regulated by the combination treatment included claudin 7, multiple caseins, xanthine oxidoreductase, and several protein synthesis, packaging, and transport genes. Genes related to structural activity including keratin 15 (morphogenesis), alpha-spectrin (cell shape via actin cytoskeleton), and chitinase-like protein 1 (tissue remodeling) were up-regulated by the combination treatment as was the transcription factor Kruppel-like factor 2 (KLF-2). However, Snail 2, which down-regulates and inhibits tight junction components, was repressed in response to the combination treatment. These results suggest coordination between endocrine and physical signals at the genomic level that produces a more specific and targeted transcriptional response associated with stage I lactogenesis. A molecular pathway analysis of the differentially expressed genes revealed that genes regulating cell signaling were linked to those regulating cell structure and adhesion.

Transforming growth factor beta1 suppresses nonmetastatic colon cancer at an early stage of tumorigenesis.

The transforming growth factor beta (TGF-beta) pathway is known to play an important role in both human and urine colon cancer. However, the staging, ligand specificity, and mechanism underlying the tumor suppressive activity of this pathway are unknown. We developed a mouse model for colon cancer that identifies an early role for TGF-beta1 in tumor suppression and implicates TGF-beta2 or TGF-beta3 in the prevention of metastasis. Analysis of the development of colon cancer in TGF-beta1 knockout mice pinpoints the defect to the hyperplasty/adenoma transition and reveals that the mechanism involves an inability to maintain epithelial tissue organization and not a loss of growth control, increased inflammatory activity, or increased genetic instability. These mice provide a unique opportunity to investigate the specific role of TGF-beta1 at this critical transition in the development of colon cancer.

Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation.

We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

Cloning and characterization of Rhizobium meliloti loci required for symbiotic root nodule invasion.

An immunological assay of root nodule polypeptides was used to analyze the nodules induced by 25 symbiotically defective Rhizobium meliloti mutants. Differences in polypeptide accumulation in these nodules were used to divide the mutants into three subsets. One subset, containing two mutant strains, was further analyzed. Nodules induced by these mutant strains lack both infection threads and bacteria. The kinetics of nodule formation by these mutant strains, by an exoB mutant, and by mixed mutant inocula suggest that the gene products required for nodule invasion may also influence nodule meristem induction. One of the two mutants characterized in this study contains a transposon Tn5 insertion in the ndvB locus, which probably results in the loss of beta-glucan synthesis. The second mutant contains a transposon in a previously uncharacterized locus. RNA analysis suggests that the newly identified locus is transcribed in free-living cultures of ndvB and exoB strains, as well as in the parental R. meliloti strain. Southern blot analysis suggests that at least a portion of this locus is duplicated. This duplication may explain the apparently leaky phenotype of the mutant strain.

Interaction of colloidal gold-labelled glucosylated albumin with endothelial cell monolayers: comparison between cryofixation and glutaraldehyde fixation.

Bovine aortic endothelial cells (BAEC) were exposed to glucosylated albumin-gold complexes (GgA), and the distribution of the tracers was compared after cryofixation and after glutaraldehyde fixation. Morphometric analysis revealed differences in the GgA distribution depending upon the method of fixation used. In BAEC monolayers cryofixed after 3 min of incubation with GgA, tracer was observed in predominately apically located vesicular elements. After 16 min of incubation, all vesicular elements were labelled, and multivesicular bodies were the prominent labelled structure. In contrast, chemically fixed monolayers exhibited a heterogeneous distribution of GgA within vesicular profiles after 3 min and 16 min of GgA incubation. The differences in tracer distribution depending upon the fixation method must be resolved before the mechanism of vesicle-mediated endothelial cell transport function is defined and universally accepted.

Parathyroid-induced angiogenesis is VEGF-dependent.

BACKGROUND: Autotransplantation of parathyroid tissue after parathyroidectomy is successful at salvaging parathyroid function. The relatively high success of parathyroid transplantation is thought to be due, in part, to the ability of parathyroid tissue to induce angiogenesis and thus recruit a new vasculature. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor produced by a number of tumors and hypoxic tissues. Using a 3-dimensional intact microvessel angiogenesis system, we evaluated the role of VEGF in the stimulation of angiogenesis by human parathyroid cells. METHODS: Freshly isolated rat microvessels embedded in a 3-dimensional collagen I matrix were treated with healthy 1-mm(3) fragments of human parathyroid tissue or isolated parathyroid cells. Other gels were supplemented with VEGF(165) or FLT-1 soluble receptor fusion protein to bind VEGF. After 11 days in culture, the gels were stained with Gs-1 lectin, a marker for rat endothelium, and linear growth of the microvessels was determined by using image analysis. Parathyroid production of VEGF was determined with reverse transcriptase-polymerase chain reaction. RESULTS: A significant increase in microvessel growth was seen in parathyroid coculture (8.4 +/- 1.0 mm) versus VEGF(165) supplemented gels (6.2 +/- 0.3 mm, P <.01). VEGF(165) significantly augmented parathyroid-stimulated angiogenesis (13.7 +/- 2.4 mm, P <.05 vs parathyroid alone). Using quantitative reverse transcriptase-polymerase chain reaction, we identified VEGF messenger RNA (mRNA) induction within 1 hour of parathyroid explant, with a 12-fold increase by 24 hours. Treatment of parathyroid cocultures with 0.2 microg/mL FLT-1 soluble receptor protein completely eliminated the parathyroid induction of angiogenesis. CONCLUSIONS: Parathyroid tissue expresses low levels of VEGF mRNA, which is significantly upregulated on explantation. Furthermore, the increased VEGF expression is essential to drive parathyroid-induced angiogenesis in our model. However, our data suggests that other parathyroid-produced factors are involved in mediating parathyroid-induced angiogenesis.

A porcine model for adipose tissue-derived endothelial cell transplantation.

The transplantation of endothelial cells represents a technology which has been suggested for applications ranging from improvement in function of implanted vascular devices to genetic therapy. The use of microvascular endothelial cell transplantation has seen increased use both in animal studies as well as clinical use. This report describes our techniques for the isolation and establishment of initial cultures of microvascular endothelial cells derived from porcine fat. A variety of anatomic sites within the pig were evaluated to determine the appropriateness of different sources of fat for endothelial cell isolation. The properitoneal fat was determined to be optimal due to the predominance of endothelium in this tissue and the ease of isolation of microvascular endothelium following collagenase digestion. The study of endothelial cell transplantation in the porcine model is now possible using the methods described for adipose tissue-derived microvessel endothelial cell isolation.

The neointima formed in endothelial cell sodded ePTFE vascular grafts results from both cellular-hyperplasia and extracellular-hypertrophy.

Endothelial cell transplantation onto polymeric vascular grafts results in the formation of a neointima. The formation of this neointima is often suggested to result from a chronic cellular hyperplasia where the terms intimal hyperplasia and intimal thickening are used interchangeably. While the formation of a midgraft neointima in sodded grafts involves a level of cell proliferation, the synthesis and deposition of extracellular matrix proteins is also a ubiquitous observation in these grafts. To assess the composition of midgraft neointima in sodded grafts, a morphometric method was developed to provide a differential quantitation of the cellular-hyperplastic and extracellular-hypertrophic elements of intimal thickening. The formed neointima on microvessel endothelial cell sodded and control (noncell-treated) ePTFE vascular grafts was quantified after 3, 12, and 52 wk of graft implantation in a canine carotid artery model. Midgraft sections of grafts were evaluated for both intimal thickness (IT) and cell density per unit volume and quantified using a PC-based image analysis program. Sodded grafts explanted at 3 wk exhibited an average neointimal cell density (3 x 10(9) cells/cm3; IT 30 microns) equivalent to cell densities observed in normal arterial media. After 12 wk the mean cell density approached a hyperplastic value (3.7 x 10(9) cells/cm3; IT 76 microns), while grafts explanted after 52 wk exhibited a mean cell density (2.8 x 10(9) cells/cm3; IT 30 microns) similar to 3-wk values. Control grafts that received no cells exhibited no midgraft cellular coverage. These results indicate that neointima formation in the midgraft region of sodded grafts occurred via mechanisms involving both a cellular hyperplasia and an extracellular hypertrophy. Differential responses occur presumably due to localized differences in cellular proliferation and cellular biosynthetic activity.

Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.

During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in an in vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fat in situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent an in vitro population of cells that accurately duplicate the in vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides an in vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in an in vitro system amenable to precise experimental manipulation.

Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell-derived vasculatures.

One of the major challenges in cell implantation therapies is to promote integration of the microcirculation between the implanted cells and the host. We used adipose-derived stromal vascular fraction (SVF) cells to vascularize a human liver cell (HepG2) implant. We hypothesized that the SVF cells would form a functional microcirculation via vascular assembly and inosculation with the host vasculature. Initially, we assessed the extent and character of neovasculatures formed by freshly isolated and cultured SVF cells and found that freshly isolated cells have a higher vascularization potential. Generation of a 3D implant containing fresh SVF and HepG2 cells formed a tissue in which HepG2 cells were entwined with a network of microvessels. Implanted HepG2 cells sequestered labeled LDL delivered by systemic intravascular injection only in SVF-vascularized implants demonstrating that SVF cell-derived vasculatures can effectively integrate with host vessels and interface with parenchymal cells to form a functional tissue mimic.

Cellular immunoisolation for islet transplantation by a novel dual porosity electrospun membrane.

Immunoisolation strategies have the potential to impact the treatment of several diseases, such as hemophilia, Parkinson's and endocrine disorders, such as parathryroid disorders and diabetes. The hallmark of these disease states is the amelioration of the disease process by replacement of the deficient protein. Naturally, several cellular therapeutic strategies like genetically modified host cells, stem cells, donor cells, or even complex tissues like pancreatic islets have been investigated. Current evidence suggests that successful strategies must incorporate considerations for local hypoxia, vascularity, and immunoisolation. Additional regulatory concerns also include safe localization of implanted therapeutic cells to allow for monitoring, dose adjustment, or removal when indicated. Local hypoxia and cellular toxicity can be detrimental to the survival of freshly implanted pancreatic islets, leading to a need for a larger initial number of islets or repeated implantation procedures. The lack of adequate donors and the large number of islet equivalents needed to achieve euglycemic states amplify the nature of this problem. We have developed a novel immunoisolation device based on electrospun nylon, primarily for islet transplantation, such that the inner component functions as a cellular barrier while allowing diffusion, whereas the outer component can be optimized for tissue integration and accelerated vascularization. Devices explanted after subcutaneous implantation in wild-type B6 mice after a period of 30 days show vascular elements in the outer layer of the electrospun device. The inner layer when intact functioned as an effective barrier to cellular infiltration. The preimplantation of such a device, with a relatively thin inner barrier membrane, will allow for adequate vascularization and reduce postimplantation hypoxia. This study demonstrates the feasibility of an electrospun isolation device that can be easily assembled, modified by varying the electrospinning parameters, and functionalized with surface-active molecules to accelerate vascularization.

Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway.

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1alpha activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

Measurement of endothelial cell migration using an improved linear migration assay. Presented at the 1995 Microcirculatory Society Meeting.

OBJECTIVE: The under-agarose migration assay developed for use with endothelial cells provides a measurement of the intrinsic migratory behavior of a cell population. However, the assay is labor intensive and lacks experimental flexibility. This migration assay has been refined and tested on human microvessel endothelial cells in the presence of a migration stimulus or on differing matrix-coated substrates. METHODS: The improved assay retains the linear geometry and mathematical basis of the under-agarose assay. Cells migrating from a cell reservoir formed with a Delrin insert are counted using an automated image-analysis system utilizing a high-contrast, fluorescent nuclear stain. From the cell counts, a stochastic measure of random migration is calculated. RESULTS: Values for random migration between the improved migration assay and the traditional under-agarose assay were very similar. Furthermore, a stochastic measure of endothelial cell migration on fibronectin was determined. CONCLUSIONS: This improved linear migration assay permits readily obtainable measures of endothelial cell migration for a number of experimental conditions. Improvements in the assay include the use of a removable fence for forming cell reservoirs, a nuclear stain to facilitate cell counting, and a more comprehensive analysis of the cell migration.

Effects of basic fibroblast growth factor on human microvessel endothelial cell migration on collagen I correlates inversely with adhesion and is cell density dependent.

Angiogenesis, new vessel growth from existing vessels, is critical to tissue development and healing. Much is known about the molecular and cellular elements of angiogenesis, such as the effects of growth factors and matrix molecules on proliferation and migration. However, it is not clear how these elements are coordinated to produce specific microvascular beds. To address this, the effects of basic fibroblast growth factor (bFGF) on beta 1 integrin-mediated adhesion relative to migration in human microvessel endothelial cells (HMVEC) was examined. Using two assays of migration that differ in the density of cells being examined, bFGF stimulated single cell migration and reduced cell migration from a confluent monolayer on collagen I. Adhesion to collagen I of HMVEC treated at low density (2-4 x 10(4) cells/cm2) with bFGF for 22 h was reduced, while bFGF increased cell adhesion of HMVEC treated at high density (6-8 x 10(4) cells/cm2). Adhesion of both bFGF-treated and untreated HMVEC was mediated by the beta 1 integrin matrix receptor. Basic FGF treatment did not significantly alter surface expression of the beta 1 integrin subunit. Reduction in bFGF-mediated adhesion correlated with delayed cell spreading and altered organization of beta 1 integrin into substrate contacts. Thus, integrin-mediated cell adhesion in microvessel endothelial cells is sensitive to regulation by a growth factor. Furthermore, the nature of the response to this signal depends on another cell regulator, cell density. In addition, modulation of cell adhesion by a growth factor may be a central regulatory feature in controlling endothelial cell migration.

Integrin-binding peptide in solution inhibits or enhances endothelial cell migration, predictably from cell adhesion.

We have examined the effects of an integrin-binding competitor, echistatin, in solution on adhesion and migration of rat microvessel endothelial cells on fibronectin in vitro. A biphasic dependence of cell motility on fibronectin surface density was observed, with a peak random motility coefficient of about 8 x 10(-9) cm2/sec occurring below 0.3 microgram/cm2 fibronectin. In the presence of echistatin at 0.5 microM, the peak random motility coefficient was similar but occurred at the significantly greater fibronectin surface density of 1.2 micrograms/cm2. Hence, the same concentration of this soluble integrin-binding competitor inhibited migration on low fibronectin densities but enhanced migration on high fibronectin densities. At the same time, echistatin decreased adhesiveness on all fibronectin surface densities. When motility was correlated explicitly with adhesiveness, a single biphasic relationship was obtained for both absence and presence of echistatin with peak motility occurring in both cases at identical adhesiveness. Both the inhibiting and enhancing effects of the soluble integrin-binding competitor on motility are predictable from its effect on adhesion, consistent with the theoretical models of Lauffenburger (15) and DiMilla et al. (3).

Transforming growth factor beta1 enhances platelet aggregation through a non-transcriptional effect on the fibrinogen receptor.

Upon activation, platelets store and release large amounts of the peptide transforming growth factor beta1 (TGFbeta1). The released TGFbeta1 can then act on nearby vascular cells to mediate subsequent vessel repair. In addition, TGFbeta1 may circulate to bone marrow and regulate megakaryocyte activity. It is not known what effect, if any, TGFbeta1 has on platelets. Adult TGFbeta1-deficient mice exhibit thrombocythemia and a mild bleeding disorder that is shown to result from faulty platelet aggregation. TGFbeta1-deficient platelets are shown to contain functional receptors, and preincubation with recombinant TGFbeta1 improves aggregation, demonstrating that TGFbeta1 plays an active role in platelet aggregation. TGFbeta1-deficient platelets fail to retain bound fibrinogen in response to aggregation agonists, but they possess normal levels of the alpha(IIb)/beta(3) fibrinogen receptor. Signaling from agonist receptors is normal because the platelets change shape, produce thromboxane A(2), and present P-selectin in response to stimulation. Consequently, activation and maintenance of alpha(IIb)/beta(3) into a fibrinogen-binding conformation is impaired in the absence of TGFbeta1. 4-Phorbol 12-myristate 13-acetate treatment and protein kinase C activity measurements suggest a defect downstream of protein kinase C in its activation cascade. Because platelets lack nuclei, these data demonstrate for the first time a non-transcriptionally mediated TGFbeta1 signaling pathway that enhances the activation and maintenance of integrin function.

HER2/neu over-expression induces endothelial cell retraction.

Over-expression of the HER2/neu (HER2) proto-oncogene in breast carcinoma imparts an enhanced metastatic potential. Metastasis requires escape of the tumor cell from the vasculature into subjacent tissue, a transmigration event across an endothelial cell (EC) monolayer. EC retraction has been reported to precede transmigration in several tumor metastatic models. Using intact human iliac vein EC monolayers, we tested the abilities of MCF-7 breast cancer cells and HER cells, a transfected MCF-7 line over-expressing HER2, to induce EC retraction. We further analyzed whether HER2 signaling influenced cancer cell-induced EC retraction. MCF-7 or HER cells were co-cultured onto mature EC monolayers. More HER than MCF-7 cells induced EC retraction (76 +/- 19% vs. 17 +/- 12%, p < 0.001) with resultant exposure of subendothelial matrix (6.80 +/- 2.86% vs. 0.85 +/- 0.39%, p < 0.001). Blockade of HER2 signaling using Herceptin nearly eliminated EC retraction (p < 0.01), while stimulation of HER2 using heregulin-beta1-augmented EC retraction (p < 0.05). Further, there was no difference between cell lines in either the number of cells adhered or the strength of adherence to EC under shear stress. These data suggest that HER2 signaling enhances metastasis in breast cancer cells by inducing EC retraction, a process that appears to precede endothelial transmigration.

Phospholamban is present in endothelial cells and modulates endothelium-dependent relaxation. Evidence from phospholamban gene-ablated mice.

Vascular endothelial cells regulate vascular smooth muscle tone through Ca2+-dependent production and release of vasoactive molecules. Phospholamban (PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Expression of PLB is reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which PLB is an important regulator of [Ca2+]i and contractility in these muscles. In the present study, we report the existence of PLB in the vascular endothelium, a nonmuscle tissue, and provide functional data on PLB regulation of vascular contractility through its actions in the endothelium. Endothelium-dependent relaxation to acetylcholine was attenuated in aorta of PLB-deficient (PLB-KO) mice compared with wild-type (WT) controls. This effect was not due to actions of nitric oxide on the smooth muscle, because sodium nitroprusside-mediated relaxation in either denuded or endothelium-intact aortas was unaffected by PLB ablation. Relative to denuded vessels, relaxation to forskolin was enhanced in WT endothelium-intact aortas. The endothelium-dependent component of this relaxation was attenuated in PLB-KO aortas. To investigate whether these changes were due to PLB, WT mouse aorta endothelial cells were isolated. Both reverse transcriptase-polymerase chain reaction and Western blot analyses revealed the presence of PLB in endothelial cells, which were shown to be >98% pure by diI-acetylated LDL uptake and nuclear counterstaining. These data indicate that PLB is present and modulates vascular function as a result of its actions in endothelial cells. The presence of PLB in endothelial cells opens new fields for investigation of Ca2+ regulatory pathways in nonmuscle cells and for modulation of endothelial-vascular interactions.