Kailin Li

Porcine kidneys as a source of ECM scaffold for kidney regeneration

OBJECTIVEnTo produce and examine decellularized kidney scaffolds from porcine as a platform for kidney regeneration research.nnnMETHODSnPorcine kidneys were decellularized with sodium dodecyl sulfate solution and Triton X-100 after the blood was rinsed. Then the renal ECM scaffolds were examined for vascular imaging, histology to investigate the vascular patency, degree of decellularization.nnnRESULTSnRenal ECM scaffolds of porcine kidneys were successfully produced. Decellularized renal scaffolds retained intact microarchitecture including the renal vasculature and essential extracellular matrix components.nnnCONCLUSIONnWe have developed an excellent decellularization method that can be used in large organs. These scaffolds maintain their basic components, and show intact vasculature system. This represents a step toward development of a transplantable organ using tissue engineering techniques.

The effective bioengineering method of implantation decellularized renal extracellular matrix scaffolds

End stage renal disease (ESRD) is a progressive loss of kidney function with a high rate of morbidity and mortality. Transplantable organs are hard to come by and hold a high risk of recipient immune rejection. We intended to establish a more effective and faster method to decellularize and recellularize the kidney scaffold for transplant and regeneration. We successfully produced renal scaffolds by decellularizing rat kidneys with 0.5% sodium dodecyl sulfate (SDS), while still preserving the extracellular matrix (ECM) 3D architecture, an intact vascular tree and biochemical components. We recellularized the kidney scaffolds with mouse embryonic stem (ES) cells that then populated and proliferated within the glomerular, vascular, and tubular structures. After in vivo implantation, these recellularized scaffolds were easily reperfused, tolerated blood pressure and produced urine with no blood leakage. Our methods can successfully decellularize and recellularize rat kidneys to produce functional renal ECM scaffolds. These scaffolds maintain their basic components, retain intact vasculature and show promise for kidney regeneration.

Platelet releasate promotes breast cancer growth and angiogenesis via VEGF–integrin cooperative signalling

Background:Selective platelet release of pro- or anti-angiogenic factors distinctly regulated angiogenesis. We hypothesised that selective release of platelet angiogenic factors could differently regulate tumour growth.Methods:Breast cancer cell proliferation, cancer cell-induced endothelial tube formation in vitro, and tumour growth in vivo were studied in the presence of protease-activated receptor 1-stimulated platelet releasate (PAR1-PR; rich in pro-angiogenic factors) or PAR4-PR (rich in anti-angiogenic factors).Results:The PAR1-PR and PAR4-PR supplementation (10%) similarly enhanced cell proliferation of MCF-7 and MDA-MB-231 breast cancer cells. The cancer cells triggered capillary-like tube formation of endothelial cells that was further enhanced by pro-angiogenic factor-rich PAR1-PR. The VEGF, but not SDF-1α, receptor blockade abolished PAR1-PR/PAR4-PR-enhanced cancer cell proliferation. Integrin blockade by RGDS had identical effects as VEGF inhibition. The Src and ERK inhibition diminished, whereas PI3K and PKC blockade abolished platelet releasate-enhanced cancer cell proliferation. Using a model of subcutaneous implantation of MDA-MB-231 cells in nude mice, PAR1-PR enhanced tumour growth more markedly than PAR4-PR, and seemed to achieve the exaggeration by promoting more profound tumour angiogenesis.Conclusions:Platelet releasate increases breast cancer cell proliferation through VEGF–integrin cooperative signalling. Pro-angiogenic factor-rich platelet releasate enhances cancer cell-induced angiogenesis more markedly, and thus exaggerates tumour growth in vivo.

The TERT promoter mutation incidence is modified by germline TERT rs2736098 and rs2736100 polymorphisms in hepatocellular carcinoma

Telomerase activation via induction of the catalytic component telomerase reverse transcriptase (TERT) plays essential roles in malignant transformation. TERT promoter-activating mutations were recently identified as a novel mechanism to activate telomerase in hepatocellular carcinoma (HCC) and many other malignancies. In addition, single nucleotide polymorphisms (SNPs) in the TERT rs2736098 and rs2736100 are significantly associated with cancer susceptibility. It is currently unclear whether different germline TERT variants modify TERT promoter mutations. Here we analyzed the TERT promoter status and genotyped the TERT SNPs at rs2736098 and rs2736100 in patients with HCC. Thirty percent of HCCs harbored TERT promoter mutations and there was a significant difference in rs2736098 and rs2736100 genotypes between wt and mutant TERT promoter-bearing HCC tumors (P = 0.007 and 0.018, respectively). For rs2736100, the cancer risk genotype CC was significantly associated with a reduced incidence of TERT promoter mutations compared to AA + AC variants [Odds ratio (OR): 0.181, 95% Confidence interval (CI): 0.0543–0.601, P = 0.004]. The rs2736098_CT genotype was significantly associated with the TERT promoter mutation-positive tumors compared to the TT genotype (OR: 5.391, 95% CI: 1.234–23.553, P = 0.025). These differences in genotype distribution did not differ between patients with a wt TERT promoter and controls. The presence of TERT promoter mutations was not associated with clinico-pathological variables. Taken together, the germline TERT genetic background may significantly affect the onset of TERT promoter mutations in HCCs, which provides a better understanding of HCC-related TERT promoter mutations and telomerase regulation in cancer.

Histone Chaperone ASF1A Predicts Poor Outcomes for Patients With Gastrointestinal Cancer and Drives Cancer Progression by Stimulating Transcription of β-Catenin Target Genes

Epigenetic mechanisms play a key role in gastrointestinal cancer (GIC) development and progression, and most studies have been focused on aberrant DNA methylation and histone modifying enzymes. However, the histone H3–H4 chaperone ASF1A is an important factor regulating chromatin assembling and gene transcription, while it is currently unclear whether ASF1A is involved in cancer pathogenesis. The present study is thus designed to address this issue. Here we showed that ASF1A expression was widespread in GIC-derived cell lines and up-regulated in primary GIC. Higher levels of ASF1A expression predicted significantly shorter patient overall survival in colorectal cancer (P = 0.0012). The further analyses of the GEO dataset validate higher ASF1A expression as a prognostic factor for CRC patients. Mechanistically, ASF1A interacted with β-catenin and promoted the transcription of β-catenin target genes including c-MYC, cyclin D1, ZEB1 and LGR5, thereby stimulating proliferation, stemness and migration/invasion of GIC cells. β-Catenin inhibition abolished these effects of ASF1A. Moreover, the ASF1A-β-catenin-ZEB1 axis down-regulated E-Cadherin expression, thereby contributing to enhanced migration/invasion of GIC cells. ASF1A over-expression and depletion facilitated and inhibited in vivo tumor growth and/or metastasis in mouse xenograft models, respectively. Taken together, ASF1A is aberrantly over-expressed in GIC tumors and plays key roles in GIC development and progression by stimulating the transcription of β-catenin target genes. ASF1A may thus be a novel target for GIC therapy and a potential prognostic marker.

Recellularization of well-preserved decellularized kidney scaffold using adipose tissue-derived stem cells.

To establish a recellularization kidney model by using adipose tissue-derived stem cells (ADSCs) as seeding cells and to investigate the growth and differentiation of ADSCs in decellularized kidney scaffolds. ADSCs were isolated using a modified method and then identified using flow cytometry analysis. Osteogenesis and adipogenesis differentiation were performed. Rat kidneys were decellularized using 0.5% sodium dodecyl sulfate. Immunofluorescence, immunohistochemistry, and scanning electron microscope were conducted to examine the scaffold microstructure. The decellularized kidney scaffold was seeded with ADSCs antegrade through the artery or retrograde through the ureter and cultured for 5-10 days. Hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry were applied to assess growth and differentiation of seeding cells within the scaffold. ADSCs populated within the glomerular, vascular, and tubular area of kidney scaffolds. Cells differentiated toward endothelial or tubular cells. Stromal cell-derived factor 1 promoted cell attachment in the scaffold. These findings suggest that ADSCs can be used as an additional new source of seeding cells within decellularized kidney scaffold. This combination may offer an alternative to donor kidney transplant. In this way, autologous ADSCs can be utilized as seeding cells in cell-scaffold kidney regeneration for further clinical transplantation.

Association Between the Telomerase rs2736098_TT Genotype and a Lower Risk of Chronic Hepatitis B and Cirrhosis in Chinese Males

Objectives:Chronic hepatitis B (CHB) is caused by infection of hepatitis B virus (HBV) and liver cirrhosis (LC) is its most common complication. The accumulated evidence indicates a genetic context of HBV infection phenotypes. Here we determine a potential association of CHB/LC with the genetic variant of telomerase reverse transcriptase (TERT), a key player in aging including immune-senescence.Methods:The study included 227 Chinese CHB patients and 315 sex/age-matched healthy controls. TERT rs2736098 and rs2736100 genotyping was performed using pre-designed TaqMan SNP genotyping assay kits. Leukocyte telomere length (LTL) was determined using quantitative PCR.Results:The rs2736098_CT/CC genotypes were significantly associated with risk of CHB compared to the TT one (OR 2.265, 95% CI 1.202–4.269, P=0.015). A similar association was also found in CHB patients with cirrhosis (CT/CC vs TT: OR 2.398, 95% CI 1.168–4.922, P=0.02). Further analyses showed that the rs2736098_TT genotype difference occurred between male controls and patients (P=0.008) and male CT/CC-carriers exhibited highly increased risk of CHB compared to male controls (CT+CC vs TT, OR 3.182, 95% CI 1.350–7.500, P=0.01). There was no difference in the rs2736100 variants between controls and CHB patients. LTL was not different between cases and controls.Conclusions:The TERT rs2736098_TT genotype is associated with a lower CHB and LC risk in Chinese males, which may have implications in CHB pathogenesis and prevention.

GABPA inhibits invasion/metastasis in papillary thyroid carcinoma by regulating DICER1 expression

The ETS family transcription factor GABPA is suggested as an oncogenic element, which is further supported by the recent reporting of it as the sole ETS member to activate the mutant TERT promoter in thyroid carcinomas (TC). However, it remains unclear how GABPA contributes to TC pathogenesis. The present study is designed to address this issue. TERT expression was significantly diminished in TERT promoter-mutated TC cells upon GABPA inhibition. Surprisingly, GABPA depletion led to robustly increased cellular invasion independently of TERT promoter mutations and TERT expression. DICER1, a component of the microRNA machinery, was identified as a downstream effector of GABPA. GABPA facilitated Dicer1 transcription while its depletion reduced Dicer1 expression. The mutation of the GABPA binding site in the DICER1 promoter led to diminished basal levels of DICER1 promoter activity and abolishment of GABPA-stimulated promoter activity as well. The forced DICER1 expression abrogated the invasiveness of GABPA-depleted TC cells. Consistently, the analyses of 93 patients with papillary thyroid carcinoma (PTC) revealed a positive correlation between GABPA and DICER1 expression. GABPA expression was negatively associated with TERT expression and promoter mutations, in contrast to published observations in cancer cell lines. Lower GABPA expression was associated with distant metastasis and shorter overall/disease-free survival in PTC patients. Similar results were obtained for PTC cases in the TCGA dataset. In addition, a positive correlation between GABPA and DICER1 expression was seen in multiple types of malignancies. Taken together, despite its stimulatory effect on the mutant TERT promoter and telomerase activation, GABPA may itself act as a tumor suppressor rather than an oncogenic factor to inhibit invasion/metastasis in TCs and be a useful predictor for patient outcomes.

Construction of engineered corpus cavernosum with primary mesenchymal stem cells in vitro

Various methods have been used to reconstruct the penis. The objective of this study was to investigate the feasibility of constructing engineered corpus cavernosum with primary mesenchymal stem cells (MSCs) in a rabbit model in vitro. Acellular corporal matrices (ACMs) were obtained from adult rabbit penile tissues through an established decellularization procedure. MSCs were separated, purified, and then seeded on ACMs to construct engineered corpus cavernosum. The seeded ACMs were subsequently cultured in an incubator for 14 days. Histological analyses showed that MSCs seeded on the ACMs had proliferated and were well distributed. Detection of CD31, vWF, smooth muscle actin (SMA), and myosin protein as well as vWF and myosin mRNA revealed that the MSCs had differentiated into endothelial cells and smooth muscle cells. In addition, cell morphology of the engineered corpus cavernosum was directly observed by transmission electron microscopy. This study demonstrated that engineered corpus cavernosum could be successfully constructed using primary MSCs in vitro. This technology represents another step towards developing engineered corpus cavernosum in vitro.

Overexpression of NEDD9 in renal cell carcinoma is associated with tumor migration and invasion

Scaffold protein neural precursor cell expressed, developmentally downregulated 9 (NEDD9) is a member of the Crk-associated substrate protein family and is known to be a biomarker in multiple cancer types. It serves a critical function in regulating cell proliferation, migration, invasion and survival. The objective of this study was to evaluate the potential effects of NEDD9 in renal cell carcinoma (RCC). The expression of NEDD9 was analyzed by immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction. NEDD9 protein and mRNA levels were significantly upregulated in RCC tissues compared with normal tissues (P<0.001). Furthermore, the NEDD9 immunostaining level was significantly associated with primary tumor stage and tumor, node, metastasis stage (P<0.05). High NEDD9 expression resulted in significantly lower survival rates for patients compared with normal NEDD9 expression (P<0.01). In addition, wound healing and transwell assays indicated that NEDD9 depletion by small interfering RNA significantly attenuated the migration and invasion of RCC cells (P<0.001). The present data suggested that NEDD9 may be a novel target for prevention and treatment of RCC metastasis and recurrence.