CD36 and macrophage scavenger receptor a modulate foam cell formation via inhibition of lipid-laden platelet phagocytosis

CD34 (+) progenitor cells are a promising source of regeneration in atherosclerosis or ischemic heart disease. However, as recently published, CD34(+) progenitor cells have the potential to differentiate not only into endothelial cells but also into foam cells upon interaction with platelets. The mechanism of platelet-induced differentiation of progenitor cells into foam cells is as yet unclear. In the present study we investigated the role of scavenger receptor (SR)-A and CD36 in platelet-induced foam cell formation. Human CD34(+) progenitor cells were freshly derived from human umbilical veins and were co-incubated with platelets (2 x 10(8)/mL) up to 14 days resulting in large lipid-laden foam cells. Developing macrophages expressed SR-A, CD36, and Lox-1 as measured by fluorescent-activated cell sorting analysis. The presence of a blocking anti-CD36 or anti-SR-A antibody nearly abrogated foam cell formation, whereas anti-Lox-1 did not affect foam cell formation. Consistently blocking either anti-CD36 or anti-SR-A antibody significantly reduced the phagocytosis of lipid-laden platelets by macrophages. We conclude that CD36 and SR-A play an important role in platelet-induced foam cell formation from CD34(+) progenitor cells and thus represent a promising target to inhibit platelet-induced foam cell formation.

Vitamin E inhibits CD36 scavenger receptor expression in hypercholesterolemic rabbits

A numerous studies suggest that Vitamin E has a preventive role in atherosclerosis, although the mechanism of action still remains unclear. CD36, a member of the scavenger receptor family is centrally involved in the uptake of oxidized low density proteins (oxLDLs) from bloodstream. During the atherosclerotic process, the lipid cargo of oxLDL accumulates in macrophages and smooth muscle cells, inducing their pathological conversion to foam cells. In the present study, we investigate the role of Vitamin E on CD36 expression in an in vivo model. Atherosclerosis was induced by a 2% cholesterol containing Vitamin E poor diet. Three groups of six rabbits each were studied. The first group (control) was fed on Vitamin E poor diet. The second group was fed with Vitamin E poor diet containing 2% cholesterol and the rabbits in the third group were fed with Vitamin E poor diet containing 2% cholesterol and received injections of 50 mg/kg of Vitamin E i.m. After 4 weeks, aortas were removed and analysed by light microscopy for atherosclerotic lesions. Aortic samples were analysed for CD36 mRNA expression. The aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by macroscopic and microscopic examination, and exhibited an increase in CD36 mRNA expression. Vitamin E fully prevented cholesterol induced atherosclerotic lesions and the induction of CD36 mRNA expression. The effects observed at the level of CD36 scavenger receptor expression in vivo suggest an involvement of reduced foam cell formation in the protective effect of Vitamin E against atherosclerosis.

T Cell Motility as Modulator of Interactions with Dendritic Cells

It is well established that the balance of costimulatory and inhibitory signals during interactions with dendritic cells (DCs) determines T cell transition from a naïve to an activated or tolerant/anergic status. Although many of these molecular interactions are well reproduced in reductionist in vitro assays, the highly dynamic motility of naïve T cells in lymphoid tissue acts as an additional lever to fine-tune their activation threshold. T cell detachment from DCs providing suboptimal stimulation allows them to search for DCs with higher levels of stimulatory signals, while storing a transient memory of short encounters. In turn, adhesion of weakly reactive T cells to DCs presenting peptides presented on major histocompatibility complex with low affinity is prevented by lipid mediators. Finally, controlled recruitment of CD8(+) T cells to cognate DC-CD4(+) T cell clusters shapes memory T cell formation and the quality of the immune response. Dynamic physiological lymphocyte motility therefore constitutes a mechanism to mitigate low avidity T cell activation and to improve the search for "optimal" DCs, while contributing to peripheral tolerance induction in the absence of inflammation.

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF(164) levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Cell-mediated BMP-2 liberation promotes bone formation in a mechanically unstable implant environment

http://www.ncbi.nlm.nih.gov/pubmed/20153849

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering

The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.

Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)-restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC-lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo

Bone formation and osseointegration of biomaterials are dependent on angiogenesis and vascularization. Angiogenic growth factors such as vascular endothelial growth factor (VEGF) were shown to promote biomaterial vascularization and enhance bone formation. However, high local concentrations of VEGF induce the formation of malformed, nonfunctional vessels. We hypothesized that a continuous delivery of low concentrations of VEGF from calcium phosphate ceramics may increase the efficacy of VEGF administration.VEGF was co-precipitated onto biphasic calcium phosphate (BCP) ceramics to achieve a sustained release of the growth factor. The co-precipitation efficacy and the release kinetics of the protein were investigated in vitro. For in vivo investigations BCP ceramics were implanted into critical size cranial defects in Balb/c mice. Angiogenesis and microvascularization were investigated over 28 days by means of intravital microscopy. The formation of new bone was determined histomorphometrically. Co-precipitation reduced the burst release of VEGF. Furthermore, a sustained, cell-mediated release of low concentrations of VEGF from BCP ceramics was mediated by resorbing osteoclasts. In vivo, sustained delivery of VEGF achieved by protein co-precipitation promoted biomaterial vascularization, osseointegration, and bone formation. Short-term release of VEGF following superficial adsorption resulted in a temporally restricted promotion of angiogenesis and did not enhance bone formation. The release kinetics of VEGF appears to be an important factor in the promotion of biomaterial vascularization and bone formation. Sustained release of VEGF increased the efficacy of VEGF delivery demonstrating that a prolonged bioavailability of low concentrations of VEGF is beneficial for bone regeneration.

VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease

Inheritance of a mutant allele of the von Hippel-Lindau tumor suppressor gene predisposes affected individuals to develop renal cysts and clear cell renal cell carcinoma. Von Hippel-Lindau gene inactivation in single renal tubular cells has indirectly been showed by immunohistochemical staining for the hypoxia-inducible factor alpha target gene product carbonic anhydrase IX. In this study we were able to show von Hippel-Lindau gene deletion in carbonic anhydrase IX positive nonneoplastic renal tubular cells, in epithelial cells lining renal cysts and in a clear cell renal cell carcinoma of a von Hippel-Lindau patient. This was carried out by means of laser confocal microscopy and immunohistochemistry in combination with fluorescence in situ hybridization. Carbonic anhydrase IX negative normal renal tubular cells carried no von Hippel-Lindau gene deletion. Furthermore, recent studies have indicated that the von Hippel-Lindau gene product is necessary for the maintenance of primary cilia stability in renal epithelial cells and that disruption of the cilia structure by von Hippel-Lindau gene inactivation induces renal cyst formation. In our study, we show a significant shortening of primary cilia in epithelial cells lining renal cysts, whereas, single tubular cells with a von Hippel-Lindau gene deletion display to a far lesser extent signs of cilia shortening. Our in vivo results support a model in which renal cysts represent precursor lesions for clear cell renal cell carcinoma and arise from single renal tubular epithelial cells owing to von Hippel-Lindau gene deletion.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.