Biomedical subjects

Yanfeng Lin

Publications and source records attributed to Yanfeng Lin.

11 recordsLinked to original sources

PubMed2006

A novel endothelial-specific heat shock protein HspA12B is required in both zebrafish development and endothelial functions in vitro.

A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and real-time PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.

Alkaline Phosphatase↗

PubMed2006

Protease activity of urokinase and tumor progression in a syngeneic mammary cancer model.

BACKGROUND: We and others have previously shown that plasminogen activators generate endogenous angiogenesis inhibitors and induce antiangiogenic activity. Here we assessed the effects of plasminogen activator overexpression on tumor progression in a syngeneic mammary cancer model. METHODS: Genes encoding murine tissue plasminogen activator (tPA), urokinase (uPA), and vector controls were stably transfected into 4T1 murine mammary cancer cells, and cell proliferation in vitro was analyzed. Cells were also implanted into female BALB/c mice (n = 12 per group), and tumor growth, lung metastases, and survival were compared. Tumor cell proliferation and microvessel formation were analyzed by immunohistochemistry using antibodies to proliferating cell nuclear antigen and CD31, respectively. 4T1 cells transfected with proteolytically inactive uPA mutants (A and B) were assayed for proliferation in vitro and tumor growth in vivo by using the same syngeneic model (eight to 10 mice per group). All statistical tests were two-sided. RESULTS: In vitro growth of uPA- and tPA-overexpressing and control 4T1 cells was similar. In vivo, however, inhibition of tumor growth and lung metastasis were inhibited in the mice carrying tPA- and uPA-overexpressing tumors, compared with controls (tumor weight at day 34: control, mean = 1760 mg, 95% confidence interval [CI] = 1434 to 2087 mg; tPA, mean = 921, 95% CI = 624 to 1217 mg; P < .001; uPA, mean = 395 mg, 95% CI = 161 to 629 mg; P < .001; number of lung metastases at day 34: control, mean = 117, 95% CI = 74 to 159; tPA, mean = 33, 95% CI = 13 to 52; uPA, mean = 15, 95% CI = 4 to 25; P < .001). Median survival was 42 (95% CI = 36 to 44), 55 (95% CI = 48 to 61), and 73 (95% CI = 51 to 86) days in the control, tPA, and uPA groups, respectively (P < .001). uPA- and tPA-expressing tumors had reduced angiogenesis and cell proliferation compared with controls. Tumors overexpressing uPA mutants grew faster than tumors expressing wild-type uPA (tumor volume at day 30: wild-type uPA, mean = 203, 95% CI = 121 to 285 mm3; control, mean = 534, 95% CI = 460 to 608 mm3; P < .001; mutant A, mean = 600, 95% CI = 520 to 679 mm3; P < .001; and mutant B, mean = 435, 95% CI = 358.9 to 511 mm3; P = .005). CONCLUSIONS: In this mouse model, uPA expression delayed tumor progression and had antiangiogenic and antiproliferative effects that may be mediated by uPA's protease activity. These results challenge the current dogma of proteases being exclusively tumor promoting and provide further rationale for exploring plasminogen activators as antitumor agents.

Animals↗

PubMed2005

Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice.

Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl-/- gonads. By E15.5, most remaining germ cells in XY Dazl-/- embryos exhibited apoptotic morphology, and XY Dazl-/- gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl-/- germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5-E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5-E13.5, shortly after its expression is first detected, and that inbred Dazl-/- mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.

Animals↗

PubMed2005

A critical role for calponin 2 in vascular development.

Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.

Amino Acid Sequence↗

PubMed2005

Magic roundabout, a tumor endothelial marker: expression and signaling.

Molecular signals that guide blood vessels to specific paths are not fully deciphered, but are thought to be similar to signals that mediate neuronal guidance. These cues are not only critical for normal blood vessel development, but may also play a major role in tumor angiogenesis. In this study, we have demonstrated the tumor endothelial specific expression of a Robo family member, magic roundabout (MRB), functionally characterized its role in endothelial cell migration and defined a signaling pathway that might mediate this function. We show that MRB is differentially over-expressed in tumor endothelial cells versus normal adult endothelial cells in numerous solid tumors. Moreover, over-expression of MRB in endothelial cells activates MRB in a ligand-independent fashion, and activation of MRB via Slit2, a putative ligand, results in inhibition of VEGF and FGF induced migration. We also demonstrate that MRB induced inhibition of endothelial migration is partially mediated by the Ras-Raf-Mek-Erk signaling pathway. We therefore hypothesize that expression of MRB is involved in regulating the migration of endothelial cells during tumor angiogenesis.

Biomarkers, Tumor↗

PubMed2005

Polyamines are involved in murine kidney development controlling expression of c-ret, E-cadherin, and Pax2/8 genes.

Polyamines play an important role in cell growth and differentiation. We studied changes in morphogenesis and the expression of the developmental control genes in the embryonic mouse kidney in response to polyamine depletion, using a kidney organ culture approach and reducing the polyamine pools with alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of ornithine decarboxylase (ODC). We found that inhibition of ODC results in a systematic kidney organogenesis phenotype, in that the DFMO-treated kidney specimens were of smaller size, had less epithelial ureteric bud branches, and their mesenchymal-derived tubule formation was retarded. These dysmorphologies were shown to be associated with changes in cell proliferation. Whole-mount in situ experiments revealed that inhibition of ODC causes increases in epithelial c-ret and E-cadherin and a decrease in mesenchymal Pax-8 expression, whereas levels of epithelial Wnt-11, mesenchymal GDNF, FoxD1, and Pax-2 transcripts remain unchanged. We studied regulation of the Pax-2 gene by analyzing a mouse line in which lacZ was driven by an 8.5 kb Pax-2 enhancer in the epithelial ureteric bud, and found that Pax-2 expression, as indicated by lacZ expression, increased after DFMO treatment. Transient transfection experiments in HEK 293 cells with the minimal Pax-2 promoter showed enhanced transcription upon reduction of the polyamine pools. We propose that ODC and polyamines have an important role in kidney organogenesis, being involved in the regulation of the expression of genes implicated in epithelial-mesenchymal tissue interactions.

Animals↗

PubMed2004

Sprouty proteins regulate ureteric branching by coordinating reciprocal epithelial Wnt11, mesenchymal Gdnf and stromal Fgf7 signalling during kidney development.

The kidney is a classic model for studying mechanisms of inductive tissue interactions associated with the epithelial branching common to many embryonic organs, but the molecular mechanisms are still poorly known. Sprouty proteins antagonize tyrosine kinases in the Egf and Fgf receptors and are candidate components of inductive signalling in the kidney as well. We have addressed the function of sprouty proteins in vivo by targeted expression of human sprouty 2 (SPRY2) in the ureteric bud, which normally expresses inductive signals and mouse sprouty 2 (Spry2). Ectopic SPRY2 expression led to postnatal death resulting from kidney failure, manifested as unilateral agenesis, lobularization of the organ or reduction in organ size because of inhibition of ureteric branching. The experimentally induced dysmorphology associated with deregulated expression of Wnt11, Gdnf and Fgf7 genes in the early stages of organogenesis indicated a crucial role for sprouty function in coordination of epithelial-mesenchymal and stromal signalling, the sites of expression of these genes. Moreover, Fgf7 induced Spry2 gene expression in vitro and led with Gdnf to a partial rescue of the SPRY2-mediated defect in ureteric branching. Remarkably, it also led to supernumerary epithelial bud formation from the Wolffian duct. Together, these data suggest that Spry genes contribute to reciprocal epithelial-mesenchymal and stromal signalling controlling ureteric branching, which involves the coordination of Ffg/Wnt11/Gdnf pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Induced repatterning of type XVIII collagen associates with ectopic Sonic hedgehog and lung surfactant C gene expression and changes in epithelial epigenesis in the ureteric bud.

How cell and tissue interactions lead to complex organ structures and differentiated cell types during organogenesis is one of the most fundamental questions in developmental biology. The embryonic lung and kidney of the mouse are useful models for studying the molecular mechanisms of morphogenesis, and in both of these organs, the epithelial bud undergoes a characteristic branching process. This review discusses the potential role of an extracellular matrix molecule, type XVIII collagen, in the generation of the branching patterns in the lung and kidney and how its experimental respecification in tissue recombinants between the ureteric bud and lung mesenchyme correlates with changes in expression of signaling molecules such as sonic hedgehog and changes in cell fate as judged by ectopic expression of the lung surfactant C gene.

Collagen Type XVIII↗

PubMed2003

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions.

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.

Animals↗

PubMed2002

Wnt-6 is expressed in the ureter bud and induces kidney tubule development in vitro.

The embryonic kidney is a classic developmental model system for studying inductive tissue interactions that govern organogenesis. We report here that Wnt-6 is expressed in the ureter bud, and that cell lines expressing Wnt-6 induce nephrogenesis in vitro. Wnt-6 cells induce tubules with similar kinetics to spinal cord (SPC) and lead to induced expression of Pax2, Pax8, Sfrp2, and E-cadherin genes, early markers of tubulogenesis. Moreover, Wnt-6 signaling rescues tubulogenesis in mesenchyme separated from Wnt-4 mutant embryos and leads to activation of Wnt-4 transcription. Wnt-6 also induces a secondary axis in early Xenopus embryos. We conclude that Wnt-6 is a candidate for the ureter epithelium-derived signal that leads to activation of kidney tubulogenesis via Wnt-4.

Animals↗

PubMed2002

Coordinating early kidney development: lessons from gene targeting.

The kidney is widely used to study the mechanisms of organogenesis. Its development involves fundamental processes, such as epithelial branching, induced morphogenesis and cytodifferentiation, which are common to the development of many other organs. Gene-targeting experiments have greatly improved our understanding of kidney development, and have revealed many important genes that regulate early kidney organogenesis, some of which have a role in inherited human kidney disorders. Although our understanding of how the kidney is assembled is still limited, these studies are beginning to provide insights into the genetic and cellular interactions that regulate early organogenesis.

Allantois↗