OBJECTIVE

To study on the first Chinese congenital thrombotic thrombocytopenic purpura (TTP) patient with respect to peripheral blood platelet count and ADAMTS13 activity, ADAMTS13 antigen, anti-endothelial cell antibody (ACEA), and thrombospondin1 (TSP1) etc. to compared it with idiopathic TTP and to explore something special and meaningful in the pathogenesis of congenital TTP.

METHODS

A total of 30 volunteers served as controls. The congenital TTP patient and 10 carriers in her pedigree as well as 7 idiopathic TTP patients were included in this study. Residue collagen binding assay and a newly developed sandwich ELISA were used for determination of ADAMTS13 activity and antigen respectively. An ELISA for ACEA and a commercial kit for TSP1 were use in the study.

RESULTS

After a "3-4 weeks regular relapse" of the congenital TTP, which is similar to that in other reports, the Chinese patient moved into a new relapse cycle. The average of the antigen levels of this patient before plasma exchange (PE) and at the interval between relapses was (22.79 +/- 14.61) U/L (P < 0.01), while that of Chinese normal control (NC) turned out to be (600.93 +/- 145.36) U/L. TSP1 detected at the same time (4.67 +/- 1.62) mg/L in the congenital TTP patient was much lower than that in NC (18.34 +/- 7.24) mg/L (P < 0.01). A value in ACEA assay was 0.58 +/- 0.06 (P < 0.01) for congenital TTP, which was a little higher than that in NC (0.40 +/- 0.13).

CONCLUSIONS

Congenital TTP may have changeable relapse cycles during episodes. ADAMTS13 antigen and TSP1 and ACEA in congenital TTP showed significant difference as compared with those in NC. ADAMTS13 and ACEA in congenital TTP were also markedly lower than those in idiopathic TTP.

We tested the hypotheses that TSP-1 participates in the initiation of remodeling of small muscular arteries in response to altered blood flow and that the N-terminal domain of TSP-1 (hepI) can reverse the pathological inward remodeling of resistance arteries from SHR.We measured (1) changes in gene/protein expression in MA of 6 week old WKY and SHR exposed to either increased (+ 100 %) or reduced blood flow (- 90 %) for 24-40 hours and (2) structural changes in MA of 12 week old SHR exposed for 3 days to hepI in organ culture.In both HF and LF of WKY, mRNA expression of eNOS, sGCα1 and PKG1β were significantly reduced (p < 0.05), whereas mRNA of TSP1 was markedly increased (p < 0.05). In MA of young SHR, similar results were obtained except that eNOS mRNA was not reduced in LF. Expression of TSP1 protein was significantly increased in LF of young WKY and SHR (p < 0.05). Exposure of MA of 12 week old SHR to hepI (1 µmol/L) resulted in a rapid lumen diameter increase (+ 12 ± 2% after 3 days) without alteration in vascular reactivity, distensibility, media surface area or cell number.These are the first observations of reduced gene expression of eNOS/sGC/PKG and increased expression of TSP1 at the initiation of arterial remodeling in young WKY and SHR, irrespective of its outward or inward outcome. Furthermore, a fragment of TSP-1 rapidly and directly reversed pathological inward arterial remodeling of SHR in vitro.

OBJECTIVE

Condylar hyperplasia (CH) is a complex disorder of the temporomandibular joint. Many studies have focused on cartilage proliferation, but the behavior of the synovium in CH is poorly understood. The aim of the present study was to investigate the expression of angiogenic-associated factors in the synovium and to discuss the possible role of the synovium in CH progression.

METHODS

CH condylar tissues were stained by hematoxylin and eosin staining, and proliferative activity was confirmed by single-photon emission computed tomography. Synovial cells isolated from the temporomandibular joint of patients with CH were collected, and flow cytometric analysis was used to examine the expression of CD34 and CD44. The gene expression of FGF-2, MMP1, MMP3, and MMP13 in synovial cells was examined by quantitative real-time polymerase chain reaction. Western blotting was used to detect the protein expression of VEGF, FGF-2, ANG1, DKK1, TSP1, MMP1, MMP3, MMP13, TIMP1, and TIMP3.

RESULTS

The typical hyperplastic area and activity were observed in condylar tissues. The expression of VEGF, FGF-2, ANG1, DKK1, TIMP1, TIMP3, and CD34 was significantly increased in the synovial cells of CH, but TSP1, MMP1, MMP3, and MMP13 expression was decreased.

CONCLUSIONS

This study exhibited a potential role for proangiogenic factors in the pathogenesis of CH.

Leukocyte-platelet rich fibrin (L-PRF) is extensively used in the dentistry field and other clinical scenarios due to its regeneration properties. The goal of the present study was to depict the L-PRF secretome and how it changes over time. We obtained L-PRF membranes and cultured them in DMEM. The secretome was collected at days 3, 7 and 21. The secretome at day 3 was analysed by LC-MS/MS and differences over time were analysed by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH). Overall, 705 proteins were identified in the secretome of L-PRF membranes after 3 days of culture, including growth factors (EGF, PDGFA) and proteins related to platelet and neutrophil degranulation. A total of 202 differentially secreted proteins were quantified by SWATH when comparing secretomes at days 3, 7 and 21. Most of them were enriched at day 3 such as MMP9, TSP1 and CO3. On the contrary, fibrinogen and CATS were found down-regulated at day 3. Growth factor and western blotting analysis corroborated the proteomic results. This is the most detailed proteome analysis of the L-PRF secretome to date. Proteins and growth factors identified, and their kinetics, provide novel information to further understand the wound healing properties of L-PRF.

TSP1 was reported to be involved in multiple biological processes including the activation of TGF-β signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17β-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia.

Keywords: CRISPR/Cas9; Nile tilapia; folliculogenesis; thrombospondin1.

Thrombospondin (TSP1) plays an important role as an antiangiogenic factor in the reproductive system of female mammals. However, its expression and function in sheep are still unclear. In the present research, the Altay sheep (a native Chinese breed) was used to analyze the expression of TSP1 in the ovary and its potential function in granulosa cells. TSP1 was widely expressed in most tissues, as shown by qPCR. In the ovary, TSP1 mRNA expression decreased during follicular to luteal growth. The TSP1 protein was expressed in a wide variety of follicles of different diameters and localized to the cytoplasm and nucleus of granulosa cells. In in vitro studies, follicle-stimulating hormone (FSH) significantly inhibited the expression of TSP1 in sheep granulosa cells. Functionally, FSH- and TSP1-specific siRNAs can promote the proliferation of sheep granulosa cells. In contrast, TSP1 mimetic peptide, ABT510, offsets the proliferation of sheep granulosa cells. Different signaling pathway inhibitors all promoted FSH-inhibited TSP1 expression, but each inhibitor had different effects on TSP1. Among them, the PI3K and ERK pathway inhibitors significantly promoted TSP1 expression and inhibited the proliferation of sheep granulosa cells.

Keywords: FSH; TSP1; cell proliferation; sheep granulosa cells.

Cynoglossus semilaevis is an important economic fish species and has long been cultivated in China. Since the completion of its genome and transcriptome sequencing, genes relating to C. semilaevis development have been extensively studied. R-spondin 3 (Rspo3) is a member of the R-spondin family. It plays an important role in biological processes such as vascular development and oncogenesis. In this study, we cloned and characterized the expression patterns and functions of C. semilaevisRspo3. Initial structural and phylogenetic analyses revealed a unique FU3 domain that exists only in ray-finned fish RSPO3. Subsequent embryonic expression profile analysis showed elevating expression of Rspo3 from gastrulation to the formation of the eye lens, while, in tail bud embryos, Rspo3 expression was significantly high in the diencephalon and mesencephalon. The overexpression of C. semilaevis Rspo3 in Danio rerio embryos resulted in a shortened rostral⁻caudal axis, edema of the pericardial cavity, stubby yolk extension, and ecchymosis. Vascular anomalies were also observed, which is consistent with Rspo3 role in vascular development. Drug treatment and a dual-luciferase reporter assay confirmed the inhibitory role of C. semilaevis Rspo3 in D. rerio Wnt/β-catenin signaling pathway. We further concluded that the FU2, FU3, and TSP1 domains regulate the maternal Wnt/β-catenin signaling pathway, while the FU1 domain regulates the zygotic Wnt/β-catenin signaling pathway. This study enriches Rspo3 research in non-model animals and serves as the basis for further research into the interactions between Rspo and the Wnt/β-catenin signaling pathway.

We investigated the 5'-flanking promoter region of the rat somatostatin receptor subtype 3 (rSSTR3). Using a cDNA probe, genomic clones containing the 5'-flanking promoter region of the rSSTR3 gene were isolated. A sequence of 5.4 kb directly upstream from the start codon was analyzed and two introns were found in the 5' untranslated region (UTR) of the cDNA sequence. The transcriptional initiation site was determined by 5' rapid amplification of cDNA ends (RACE), primer extension and RNase protection analysis with cerebellar RNA. Two major transcriptional initiation sites were found at position 1040 (tsp1) and -856 (tsp2) relative to the translational initiation site. Like a number of other promoters of G-protein-coupled receptors, the rSSTR3 gene lacks TATA and CAAT motifs and includes G+C-rich regions. Functional analysis of the promoter region by transfecting rSSTR3 luciferase-reporter gene constructs into rat pituitary GH3 cells and HEK 293 cells indicated that a 107-bp region upstream of tsp2 was sufficient to drive transcription. Furthermore a 562-bp region at position -1304 to -1865 upstream of the ATG start codon exerted a negative regulatory effect on transcriptional activity.

The complement components C8α and C8β mediate the formation of the membrane attack complex (MAC) to resist pathogenic bacteria and play important roles in innate immunity. Full-length complement C8α (Pv-C8α) and C8β (Pv-C8β) cDNA were identified in the darkbarbel catfish Pelteobagrus vachellii, and their mRNA expression levels were analyzed after ammonia-N and pathogen treatment. The Pv-C8α gene contained 1983 bp, including a 1794-bp open reading frame (ORF) encoding 598 amino acids. The Pv-C8β gene contained 1952 bp, including a 1761-bp ORF encoding 587 amino acids. Pv-C8α and Pv-C8β had the highest amino acid identity with rainbow trout Oncorhynchus mykiss C8α (62%) and Japanese flounder Paralichthys olivaceus C8β (83%), respectively. Sequence analysis indicated that both Pv-C8α and Pv-C8β contained a thrombospondin type-1 (TSP1) domain, a low-density lipoprotein receptor class A (LDLR-A) domain, a membrane attack complex/perforin (MACPF) domain and an epidermal growth factor-like (EGF-like) domain. In addition, Pv-C8α and Pv-C8β were mainly distributed in the liver, head kidney, spleen, and eggs. Under ammonia-N stress, the Pv-C8α and Pv-C8β mRNA levels significantly decreased (P < 0.05), with minimum levels, respectively, attained at 24 and 48 h in the liver, 48 and 24 h in the head kidney, and 24 and 24 h in the spleen. After Aeromonas hydrophila challenge, the Pv-C8α and Pv-C8β mRNA levels significantly increased (P < 0.05), with maximum levels, respectively, attained at 48 and 24 h in the liver, 24 and 48 h in the head kidney, and 48 and 48 h in the spleen. The present study indicated that Pv-C8α and Pv-C8β exhibited important immune responses to infection and that ammonia-N in water decreased the immune responses of Pv-C8α and Pv-C8β.

The integrin family receptors stimulate the cellular proliferation and migration through the focal adhesion pathway by the activation of PTK2, VASP and TSP1 proteins. The purpose of this study was to investigate the integrin-ligated motifs through the activation of focal adhesion pathway. A chimeric peptide was predicted from the integrin-mediated ligands by bioinformatics tools. The VSMCs were treated with the chimeric peptide and simvastatin. The PTK2, VASP and TSP1 protein and gene expression levels were measured by RT-qPCR and Western Blotting techniques, respectively. AutoDock Tools were used for the docking technique. The PTK2, VASP and TSP1 protein expression levels increased significantly in the VSMCs treated with chimeric peptide in conversely with the effects of simvastatin. The docking results suggested two motifs in the chimeric peptide. In conclusion, the chimeric peptide activated the focal adhesion pathway. The motifs 1 and 2 may be directly involved in the transduction of signal by integrin family receptors.

Sexual communication between complementary mating partners in the fungal group of zygomycetes is mediated by the trisporoid pheromone system. A key enzyme towards biosynthesis of hormonally active trisporoids is 4-dihydromethyltrisporate dehydrogenase (TSP1), an enzyme occurring in all zygomycetous fungi. Trisporic acid and some of its precursor molecules serve as pheromones for recognizing complementary mating partners and for induction of the differentiation program towards sexual spore formation. In the parasitic zygomycete Parasitella parasitica, a biotrophic fusion parasite infecting many other zygomycetes, these substances have an additional function: They are also responsible for host-parasite recognition and the formation of the characteristic infection structures. Parasitic interactions are mating type dependent as well. In the Mucor-related mycoparasite P. parasitica we can study both types of communication in parallel. We were interested in protein structures of TSP1 from P. parasitica, the genome of which was recently sequenced by us, and especially in the mechanisms involved in the switch from sexual to parasitic communication. P. parasitica contains at least six genes coding for TSP1-like proteins. We created models of tertiary structures and performed protein-protein docking with the resulting protein structures to simulate dimerization and to provide support for probable regulatory mechanisms at the protein level. The resulting structure models show differences in putative activity and binding preferences between the different TSP1-like proteins. Two of them seem to be able to form solid binding pockets for substrate and cosubstrate after dimerization. The other four TSP1-like proteins are more likely to represent regulating subunits for the two active isoforms. The ability to form homodimers with enzymatic activity could be the crucial difference between sexual and parasitic communication pathways. TSP1 PARPA_07791 forms enzymatically inactive homodimers. The second TSP1, PARPA_04105, forms active homodimers and could be responsible for the parasitic pathway of communication. Both TSP1 proteins can form more or less active heterodimers with the additional TSP1-like proteins. TSP1 PARPA_07791 mediates the sexual pathway probably as in other zygomycetous fungi like Mucor mucedo. High sequence identities between this TSP1 isomer and TSP1 proteins from other zygomycetes substantiate its function. This bioinformatic study supports previous experimental findings of post-translational regulation of 4-dihydromethyltrisporate dehydrogenases in zygomycetes and, for the first time, provides a substantiated hypothesis of the underlying mechanism.

Stroke is a multi-factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 106 human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q-PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation-specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper-methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo-methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.

13C spin-lattice relaxation times in the laboratory frame, ranging from 1.4 to 36 h, have been measured on a suite of five natural type Ia and Ib diamonds at 4.7 T and 300 K. Each of the diamonds contains two types of fixed paramagnetic centers with overlapping inhomogeneous electron paramagnetic resonance (EPR) lines. EPR techniques have been employed to identify these defects and to determine their concentrations and relaxation times at X-band. Spin-lattice relaxation behavior of 13C in diamonds containing paramagnetic P1, P2, N2. and N3 centers are discussed. Depending on the paramagnetic impurity types and concentrations present in each diamond, three different nuclear spin-lattice relaxation (SLR) paths exist, namely that due to electron SLR mechanisms and two types of three-spin processes (TSPs). The one three-spin process (TSP1) involves a simultaneous transition of two electron spins belonging to the same hyperfine EPR line and a flip of a 13C spin, while the other process (TSP2) involves two electron spins belonging to different hyperfine EPR lines and a 13C spin. It is shown that the thermal contact between the 13C nuclear Zeeman and electron dipole-dipole interaction reservoirs is field dependent, thus forming a bottleneck in the 13C relaxation path due to TSP1 at high magnetic fields.

BACKGROUND

Low-dose metronomic chemotherapy (LDMC) is an alternative for treatment of patients with late-stage prostate cancer (PC) not susceptible to regular chemotherapy due to its severe side effects. The exact working mechanisms of LDMC have not been established, although anti-angiogenic effects have been identified. In PC, several studies show clinical effects from LDMC but the mode of action and the role of androgen signaling for its effect are not known. In this study, we used a xenograft model to evaluate the effect of LDMC on PC growth in relation to androgen deprivation.

METHODS

Subcutaneous human castration-resistant PC xenografts were treated with LDMC using cyclophosphamide (CPA). Treatment effect was compared to treatment with maximum tolerated dose (MTD) and also between intact and castrated mice. Microvessel density (MVD), and factors important for angiogenesis were analyzed with immunohistochemistry and real-time-PCR.

RESULTS

Tumors treated with LDMC were 50% smaller than untreated controls. Tumors in non-castrated mice were not affected by LDMC, but in an androgen receptor (AR) negative tumor model, tumor inhibiting effect were seen in both intact and castrated animals, indicating mechanism via AR. MTD resulted in similar growth inhibition as LDMC in castrated mice, but resulted in severe weight loss. Despite that LDMC induced TSP1 mRNA expression, and the hypoxic area in the tumors was slightly increased, no decrease in MVD was detected.

CONCLUSIONS

This study shows that a low-dose metronomic scheduling of CPA was as efficient as MTD treatment, and resulted in fewer side effects. It also demonstrates that a functional androgen signaling axis inhibits this effect despite the castration-resistance of the tumor cells. The anti-angiogenic nature of the effect of LDMC could not be confirmed and further studies to elucidate the working mechanism for treatment response are needed.

The endothelial nitric oxide synthase (eNOS) signaling pathway in endothelial cells has multiple physiological significances. It produces nitric oxide (NO), an important vasodilator, and enables a long-term proliferative response, contributing to angiogenesis. This signaling pathway is mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic species that is often targeted to inhibit tumor angiogenesis. However, inhibiting VEGF-mediated eNOS signaling can lead to complications such as hypertension. Therefore, it is important to understand the dynamics of eNOS signaling in the context of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important angiogenic inhibitor that, through interaction with its receptor CD47, has been shown to redundantly inhibit eNOS signaling. However, the exact mechanisms of TSP1's inhibitory effects on this pathway remain unclear. To address this knowledge gap, we established a molecular-detailed mechanistic model to describe VEGF-mediated eNOS signaling, and we used the model to identify the potential intracellular targets of TSP1. In addition, we applied the predictive model to investigate the effects of several approaches to selectively target eNOS signaling in cells experiencing high VEGF levels present in the tumor microenvironment. This work generates insights for pharmacologic targets and therapeutic strategies to inhibit tumor angiogenesis signaling while avoiding potential side effects in normal vasoregulation.

Rho-kinase (ROCK) inhibitors, a novel class of anti-glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin-1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine-serine-lysine-leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose-dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.

Keywords: ROCK inhibitor; glaucoma; outflow facility; thrombospondin-1; trabecular meshwork.

Inherited retinal diseases such as retinitis pigmentosa (RP) can be caused by thousands of different mutations, a small number of which have been successfully treated with gene replacement. However, this approach has yet to scale and may not be feasible in many cases, highlighting the need for interventions that could benefit more patients. Here, we found that microglial phagocytosis is upregulated during cone degeneration in RP, suggesting that expression of "don't eat me" signals such as CD47 might confer protection to cones. To test this, we delivered an adeno-associated viral (AAV) vector expressing CD47 on cones, which promoted cone survival in three mouse models of RP and preserved visual function. Cone rescue with CD47 required a known interacting protein, signal regulatory protein alpha (SIRPα), but not an alternative interacting protein, thrombospondin-1 (TSP1). Despite the correlation between increased microglial phagocytosis and cone death, microglia were dispensable for the pro-survival activity of CD47, suggesting that CD47 interacts with SIRPα on non-microglial cells to alleviate degeneration. These findings establish augmentation of CD47-SIRPα signaling as a potential treatment strategy for RP and possibly other forms of neurodegeneration.

Keywords: Gene therapy; Neurodegeneration; Ophthalmology; Therapeutics.

To clinically characterize a cohort of patients with ectopia lentis (EL), or Marfanoid features in whom a definite genetic diagnosis of Marfan syndrome (MFS) had been excluded (atypical MFS), and to evaluate the contribution of mutations in ADAMTSL4 (OMIM * 610113), and P3H2 (LEPREL1; OMIM * 610341) to disease in this population.

Subjects underwent comprehensive ophthalmic examination, including keratometry. Mutational analysis of ADAMTSL4 and P3H2 was undertaken using PCR, high resolution melting analysis, and sequencing. The frequency of c.2237G>A; p.(Arg746His) was determined in an unaffected Polynesian cohort. Haplotype analysis used tagged single nucleotide polymorphic markers.

Mutational analysis of ADAMTSL4 identified two pathogenic variants in ADAMTSL4 in 11/31 (35%) probands, consistent with the autosomal recessive EL phenotype. A recurrent, rare missense variant in ADAMTSL4, c.2237G>A; p.(Arg746His), was present in 10 probands -(8 homozygotes), predominantly of Polynesian descent, and all shared the same haplotype. p.(Arg746His) affects the Thrombospondin1 (TSP1) domain of the protein and is predicted to be pathogenic. No pathogenic variants in P3H2 were identified.

A recurrent pathogenic ADAMTSL4 variant is a major cause of early onset autosomal recessive EL in a Cook Island Māori population and associated with a common haplotype, suggesting a founder effect. Children presenting under the age of 5 years, particularly of Cook Island or New Zealand Māori descent, with isolated ectopia lentis, should in the first instance be tested for this single variant.

Background: Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established.

Methods: Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92 ^endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated.

Results: miR-17, -18a, -20a, -19b, and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92 ^endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92 ^endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92 ^endo-/- after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate.

Conclusions: These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.

Keywords: acute kidney injury; microRNA; renal microvasculature.

With a well-understood function in mammals, R-spondin1 (Rspo1) is an important regulator of ovarian development via the Wnt/β-catenin pathway. Rspo1 deficiency causes retardation of ovarian development in XX fish, and increases Rspo1 function induces femininity and sex reversal in XY fish. In this study, Rspo1 was successfully cloned from loach (Misgurnus anguillicaudatus), and its expression profile was analyzed. The full-length cDNA of Misgurnus anguillicaudatus Rspo1 (MaRspo1) comprised 1322 bp and included an open reading frame (ORF) of 795 bp, which encoded a predicted polypeptide measuring 264 amino acids in length. Phylogenetic and gene structure analyses showed a highly conserved sequence of MaRspo1 (identical to the Rspo1 genes of other species), consisting of an N-terminal signal peptide (SP), two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat (TSP1) and a C-terminal region. Real-time PCR revealed the female-biased expression profile of MaRspo1, with the highest expression level among tested tissues detected in ovary. Investigation of MaRspo1 expression levels throughout the early development stage (10-60 days post hatching) under three temperature treatments (25 °C, 28 °C, and 31 °C) revealed significantly differential expression of MaRspo1 among the three temperature groups, with decreased MaRspo1 expression in the high-temperature (31 °C) group. The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of MaRspo1 in both females and males. Taken together, the results of this study provide the basis for the further investigation of the molecular mechanism of Rspo1 in response to temperature.

Keywords: DNA methylation; High temperature; Misgurnus anguillicaudatus; R-spondin1; Sex differentiation.

Objective: Increasing evidence indicates an interaction between the synovium and the cartilage in the temporomandibular joint (TMJ) and other joints. We recently demonstrated that the expression of proangiogenic factors was enhanced and that of factors promoting matrix degradation was decreased in synovial fibroblasts in condylar hyperplasia (CH). The aim of this study was to explore whether CH chondrocytes can affect the expression of these factors of synovial fibroblasts in a co-culture system.

Study design: The expressions of vascular endothelial growth factor (VEGF), cluster of differentiation 34 (CD34), fibroblast growth factor 2 (FGF-2), and tissue inhibitor of metalloproteinase 1 (TIMP1) from CH condylar tissues were observed by using immunohistochemical methods. Synovial fibroblasts of control tissues were co-cultured with the chondrocytes of CH, and protein expressions of VEGF, FGF-2, thrombospondin 1 (TSP1), matrix metalloproteinase 3 (MMP3), and TIMP1 were examined by using Western blotting.

Results: Positive staining for VEGF, CD34, FGF-2, and TIMP1 was found in the hypertrophic cartilage layer of CH condylar tissues. Protein expressions of VEGF, FGF-2, and TIMP1 were significantly increased in co-cultured synovial fibroblasts, but TSP1 and MMP3 expressions were decreased.

Conclusions: The angiogenic factors and matrix degradation-related factors in synovial fibroblasts co-cultured with CH chondrocytes showed the same trends as those in synovial fibroblasts from CH tissue, suggesting potential cross-talk between synovial fibroblasts and chondrocytes during CH progression.

Osteosarcomas, especially those with metastatic or unresectable disease, have limited treatment options. The antitumoreffects of pharmacologic inhibitors of angiogenesis in osteosarcomas are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here, we demonstrated that thrombospondin 1 (TSP1) is a potent inhibitor of osteosarcoma cells MG-63 growth and metastasis. Moreover, we demonstrate up-regulation of TSP-1 facilitated expression of vasculostatin in MG-63 cells. In angiogenesis assays, over-expression of TSP-1 inhibited MG-63 cells induced tube formation of human umbilical vein endothelial cells (HUVECs) and this effect required CD36. Finally, in xenografted tumors, we observed that TSP1 overexpression inhibited angiogenesis and tumor growth. These results provided the strong evidence for an important role of the TSP-1/CD36/vasculostatin signaling axis in mediating the anti-angiogenic activity of osteosarcoma.