Proangiogenic Properties of Thrombospondin-4.

OBJECTIVE: Thrombospondin-4 (TSP-4) is 1 of the 5 members of the thrombospondin protein family. TSP-1 and TSP-2 are potent antiangiogenic proteins. However, angiogenic properties of the 3 other TSPs, which do not contain the domains associated with the antiangiogeneic activity of TSP-1 and TSP-2, have not been explored. In our previous studies, we found that TSP-4 is expressed in the vascular matrix of blood vessels of various sizes and is especially abundant in capillaries. We sought to identify the function of TSP-4 in the regulation of angiogenesis. APPROACH AND RESULTS: The effect of TSP-4 in in vivo angiogenesis models and its effect on angiogenesis-related properties in cultured cells were assessed using Thbs4(-/-) mice, endothelial cells (EC) derived from these mice, and recombinant TSP-4. Angiogenesis was decreased in Thbs4(-/-) mice compared with wild-type mice. TSP-4 was detected in the lumen of the growing blood vessels. Mice expressing the P387 TSP-4 variant, which was previously associated with coronary artery disease and found to be more active in its cellular interactions, displayed greater angiogenesis compared with A387 form. Lung EC from Thbs4(-/-) mice exhibited decreased adhesion, migration, and proliferation capacities compared with EC from wild-type mice. Recombinant TSP-4 promoted proliferation and the migration of EC. Integrin α2 and gabapentin receptor α2δ-1 were identified as receptors involved in regulation of EC adhesion, migration, and proliferation by TSP-4. CONCLUSION: TSP-4, an extracellular matrix protein previously associated with tissue remodeling, is now demonstrated to possess proangiogenic activity.

Control of organization and function of muscle and tendon by thrombospondin-4.

Thrombospondins (TSPs) are multifunctional proteins that are deposited in the extracellular matrix where they directly affect the function of vascular and other cell types. TSP-4, one of the 5 TSP family members, is expressed abundantly in tendon and muscle. We have examined the effect of TSP-4 deficiency on tendon collagen and skeletal muscle morphology and function. In Thbs4(-/-) mice, tendon collagen fibrils are significantly larger than in wild-type mice, and there is no compensatory over-expression of TSP-3 and TSP-5, the two TSPs most highly homologous to TSP-4, in the deficient mice. TSP-4 is expressed in skeletal muscle, and higher levels of TSP-4 protein are associated with the microvasculature of red skeletal muscle with high oxidative metabolism. Lack of TSP-4 in medial soleus, red skeletal muscle with predominant oxidative metabolism, is associated with decreased levels of several specific glycosaminoglycan modifications, decreased expression of a TGFß receptor beta-glycan, decreased activity of lipoprotein lipase, which associates with vascular cell surfaces by binding to glycosaminoglycans, and decreased uptake of VLDL. The soleus muscle is smaller and hind- and fore-limb grip strength is reduced in Thbs4(-/-) mice compared to wild-type mice. These observations suggest that TSP-4 regulates the composition of the ECM at major sites of its deposition, tendon and muscle, and the absence of TSP-4 alters the organization, composition and physiological functions of these tissues.

Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload.

Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4(-/-)) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4(-/-) mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4(-/-) mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4(-/-) mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling.

Thrombospondin-4 regulates vascular inflammation and atherogenesis.

RATIONALE: Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains unknown. OBJECTIVE: We have examined the effects of TSP-4 gene (Thbs4) knockout on the development of atherosclerotic lesions in ApoE(-/-) mice. METHODS AND RESULTS: Deficiency in TSP-4 reduced atherosclerotic lesions: at 20 weeks of age, the size of the aortic root lesions in Thbs4(-/-)/ApoE(-/-) mice was decreased by 48% in females and by 39% in males on chow diets; in mice on Western diets, lesions in the descending aorta were reduced by 30% in females and 33% in males. In ApoE(-/-) mice, TSP-4 was abundant in vessel areas prone to lesion development and in the matrix of the lesions themselves. TSP-4 deficiency reduced the number of macrophages in lesions in all groups by ≥ 2-fold. In addition, TSP-4 deficiency reduced endothelial cell activation (expression of surface adhesion molecules) and other markers of inflammation in the vascular wall (decreased production of monocyte chemoattractant protein-1 and activation of p38). In vitro, both the adhesion and migration of wild-type macrophages increased in the presence of purified recombinant TSP-4 in a dose-dependent manner (up to 7- and 4.7-fold, respectively). These responses led to p38-MAPkinase activation and were dependent on ß(2) and ß(3) integrins, which recognize TSP-4 as a ligand. CONCLUSIONS: TSP-4 is abundant in atherosclerotic lesions and in areas prone to development of lesions and may influence the recruitment of macrophages by activating endothelial cells and directly interacting with macrophages to increase their adhesion and migration. Our observations suggest an important role for this matricellular protein in the local regulation of inflammation associated with atherogenesis.

The minimal instrumentation requirements for Hoechst side population analysis: stem cell analysis on low-cost flow cytometry platforms.

The Hoechst side population (SP) technique is a critical method of identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues. In this technique, the cell-permeable DNA-binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells and early progenitors subsequently pump this dye out via an ATP-binding cassette membrane pump-dependent mechanism, resulting in a low-fluorescence "tail" (the SP) when the cells are analyzed by flow cytometry. This population contains stem cells and early progenitors. One significant drawback of this method is the requirement of an UV laser to excite the Hoechst 33342. Unfortunately, flow cytometers equipped with UV sources are expensive to own and operate and are not readily available to many laboratories or institutions. In the interests of designing a less expensive flow cytometric system for stem cell analysis, we determined the minimum UV excitation and instrumentation requirements for measuring Hoechst SP. Less than 3 mW of UV laser output was required for adequate resolution of Hoechst SP on two cuvette-based flow cytometers, one of which was a simple, inexpensive benchtop analyzer (the Quanta Analyzer; NPE Systems). Furthermore, Hoechst SP could also be adequately resolved on this epifluorescence-based cytometer platform using two nonlaser UV sources, a mercury arc lamp with a UV bandpass filter and a UV-emitting light-emitting diode. These results suggest that an economical flow cytometric system can be designed that is capable of resolving Hoechst SP, with a cost far lower than most UV laser-equipped commercial systems. An inexpensive system of this type would make Hoechst SP analysis available to a much broader group of stem cell investigators.

Discrimination of the Hoechst side population in mouse bone marrow with violet and near-ultraviolet laser diodes.

BACKGROUND: Discrimination of stem cells with flow cytometric analysis of Hoechst 33342 efflux by the ABCG2 transporter (termed the Hoechst side population, or SP technique) is a valuable methodology for identifying bone marrow progenitors enriched with stem cells. Unfortunately, it requires a ultraviolet (UV) laser source, usually necessitating an expensive and maintenance-intensive argon- or krypton-ion gas laser on a large-scale cell sorter. In this study, we evaluated the ability of recently available violet and near-UV laser diodes to discriminate Hoechst SP on smaller cuvette-based flow cytometers. METHODS: Violet laser diodes (emitting at 408 and 401 nm) and a near-UV laser diode (emitting at 370 nm) were mounted on a BD Biosciences LSR II and evaluated for their ability to discriminate Hoechst SP in murine bone marrow. RESULTS: The violet laser diodes discriminated the Hoechst SP, but with poorer resolution than with the standard UV gas laser on a large-scale cell sorter. The near-UV laser diode, in contrast, gave excellent Hoechst SP resolution. CONCLUSIONS: These evaluations indicated that near-UV laser diodes give excellent Hoechst SP resolution on cuvette-based instruments. As the next generation of cell sorters integrate cuvette-based cell interrogation into conventional jet-in-air cell separation, these laser sources should become applicable for analysis and physical separation of Hoechst SP cells.

Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice.

Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes. We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos. A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death. This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication. These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.

Imprint control element-mediated secondary methylation imprints at the Igf2/H19 locus.

Understanding the molecular basis of monoallelic expression as observed at imprinted loci is helpful in understanding the mechanisms underlying epigenetic regulation. Genomic imprinting begins during gametogenesis with the establishment of epigenetic marks on the chromosomes such that paternal and maternal chromosomes are rendered distinct. During embryonic development, the primary imprint can lead to generation of secondary epigenetic modifications (secondary imprints) of the chromosomes. Eventually, either the primary imprints or the secondary imprints interfere with transcription, leading to parent-of-origin-dependent silencing of one of the two alleles. Here we investigated several aspects pertaining to the generation and functional necessity of secondary methylation imprints at the Igf2/H19 locus. At the H19 locus, these secondary imprints are, in fact, the signals mediating paternal chromosome-specific silencing of that gene. We first demonstrated that the H19 secondary methylation imprints are entirely stable through multiple cell divisions, even in the absence of the primary imprint. Second, we generated mouse mutations to determine which DNA sequences are important in mediating establishment and maintenance of the silent state of the paternal H19 allele. Finally, we analyzed the dependence of the methylation of Igf2DMR1 region on the primary methylation imprint about 90 kilobases away.

Determination of T and B Cell Epitopes of Aspergillus fumigatus Ribotoxin and Heat Shock Protein.

Ribotoxin Asp f1 and heat shock protein Asp hsp1 from Aspergillus fumigatus represent the major components of A. fumigatus allergic complex. Eight computer predicted peptides corresponding to probable T epitopes of Asp f1 (4 peptides) and Asp hsp1 (4 peptides) have been synthesized according to the primary sequence of the proteins. The peptides Asp f1 15-27, 87-99 and Asp hsp 84-96, able to bind high affinity sera from mice BALB/c immunized with crude A. fumigatus extract, were considered to represent B cell epitopes. To screen T cell epitopes among the peptides splenocytes from mice immunized with crude A. fumigatus extract were stimulated in vitro with A. fumigatus or peptides, and their Ab production was analyzed. Spontaneous A. fumigatus-specific Ab production was found in unstimulated control. In cultures stimulated with 20 &mgr;g/ml of A. fumigatus preparation Ab production was blocked by 92%. The peptide Asp f1 87-99 decreased synthesis of A. fumigatus-specific Abs by 80%. The peptide Asp f1 15-27 and those overlapping hsp1 18-31 and 22-36 induced 34-37% decrease in Ab production. Other A. fumigatus-derived or irrelevant peptides did not affect specific Ab production. Thus, these peptides, containing T cell epitope, may be considered as potential candidates for peptide-based immunotherapy of allergic aspergillosis.