The role of p73 in cancer – a pathway towards more effective cancer therapy

The p53-family consists of three transcription factors, p53, p73 and p63. The family members have similar but also individual functions connected to cell cycle regulation, development and tumorigenesis. p53 and p73 act mainly as tumor suppressors. During DNA damage caused by anticancer drugs or irradiation, p53 and p73 levels are upregulated in cancer cells leading to apoptosis and cell cycle arrest. p53 is mutated in almost 50 per cent of the cancers, causing the cancer cells unable to undergo cell death. Instead, p73 is rarely mutated in cancer cells and because of that could be more viable target for anticancer therapy. The network surrounding the regulation of p73 is extensive and has several potential targets for cancer therapy. One of the most studied is Itch ligase, the negative regulator of p73 levels. Gene therapy directed towards knockdown of Itch ligase is a potential approach but in need for more in vivo proof. p73 has two isoforms, transactivating TA-forms and dominant-negative ΔN-forms. The specific regulation of these isoforms could also offer a possible way for more effective cancer treatment. The literature work includes information of structures, isoforms, functions and possible therapeutic targets of p73. Also the main therapeutic approaches to date are introduced. The experimental part is based on transfection and cytotoxicity studies done e.g. in pancreatic cancer cells (Mia PaCa-2, PANC1, BxPc-3 and HPAC). The aim of the experimental work was to optimize the conditions for effective transfection with DAB16 dendrimer nanoparticles and to measure the cytotoxicity of plain dendrimers and DAB16-pDNA complexes. Also the protein levels of p73 and Itch ligase were measured by Western blotting. The work was done as a part of a bigger project, which was aiming to down regulate Itch ligase (negative regulator of p73) by siRNA/shRNA. Tranfection results were promising, showing good transfection efficacy with DAB16 N/P30 in pancreatic cancer cells (except in BxPc-3). Pancreatic cancer cells showed recovery in 3 days after they were exposed to plain dendrimer solution or to DAB16-pDNA. Measurement of protein levels by Western blotting was not optimal and the proposals for the improvement regarding e.g. the gels and the extracted protein amounts have been done.

Angelman Syndrome Protein UBE3A Interacts with Primary Microcephaly Protein ASPM, Localizes to Centrosomes and Regulates Chromosome Segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

Cytochrome P4504f, a potential therapeutic target limiting neuroinflammation

Inflammatory processes are involved in the pathogenesis and/or progression of acute central nervous system (CNS) infection, traumatic brain injury and neurodegenerative disorders among others indicating the need for novel strategies to limit neuroinflammation. Eicosanoids including leukotrienes, particularly leukotriene B-4 (LTB4) are principle mediator(s) of inflammatory response, initiating and amplifying the generation of cytokines and chemokines. Cytochrome P450 (Cyp), a family of heme proteins mediate metabolism of xenobiotics and endogenous compounds, such as eicosanoids and leukotrienes. Cytochrome P4504F (Cyp4f) subfamily includes five functional enzymes in mouse. We cloned and expressed the mouse Cyp4f enzymes, assayed their relative expression in brain and examined their ability to hydroxylate the inflammatory cascade prompt LTB4 to its inactive 20-hydroxylated product. We then examined the role of Cyp4fs in regulating inflammatory response in vitro, in microglial cells and in vivo, in mouse brain using lipopolysacharide (LPS), as a model compound to generate inflammatory response. We demonstrate that mouse brain Cyp4fs are expressed ubiquitously in several cell types in the brain, including neurons and microglia, and modulate inflammatory response triggered by LPS, in vivo and in microglial cells, in vitro through metabolism of LTB4 to the inactive 20-hydroxy LTB4. Chemical inhibitor or shRNA to Cyp4fs enhance and inducer of Cyp4fs attenuates inflammatory response. Further, induction of Cyp4f expression lowers LTB4 levels and affords neuroprotection in microglial cells or mice exposed to LPS. Thus, catalytic activity of Cyp4fs is a novel target for modulating neuroinflammation through hydroxylation of LTB4. (C) 2011 Elsevier Inc. All rights reserved.

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma Laboratory investigation

Object. Insulin-like growth factor binding proteins (IGEBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. Methods. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. Results. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. Conclusions. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Design, synthesis, and biological activity of rhodium metalloinsertors

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.

Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.

In addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.

To explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.

Finally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both Δ and Λ enantiomers of [Rh(chrysi)(phen)(DPE)]²⁺ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.

Differential activity of GSK-3 isoforms regulates NF-kappa B and TRAIL- or TNF alpha induced apoptosis in pancreatic cancer cells

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3 alpha or GSK-3 beta. In contrast, depletion of GSK-3 beta, but not GSK-3 alpha, sensitized PDA cell lines to TNF alpha-induced cell death. Further experiments demonstrated that TNF alpha-stimulated I kappa B alpha phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3 beta-deficient MEFs. Nonetheless, inhibition of GSK-3 beta function in MEFs or PDA cell lines impaired the expression of the NF-kappa B target genes Bcl-xL and cIAP2, but not I kappa B alpha. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3 beta targeted to the nucleus but not GSK-3 beta targeted to the cytoplasm, suggesting that GSK-3 beta regulates NF-kappa B function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3 beta overexpression and nuclear localization contribute to TNF alpha and TRAIL resistance via anti-apoptotic NF-kappa B genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Porównawcza analiza udziału pokrewnych kinaz MAPK (MKK1, MKK2, MKK4 i MKK5) Arabidopsis thaliana w odpowiedzi na stres suszy

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.