Padma Murthi

Effect of angiotensin II type 1 receptor blockade on experimental hepatic fibrogenesis.

BACKGROUND/AIMS The aim of this study was to investigate whether in the liver, as in other tissues, there is evidence that angiotensin II, acting via the angiotensin II type 1 receptor (AT1-R), plays a role in fibrogenesis. METHODS Sprague-Dawley rats were divided into three groups; sham, bile duct ligated (BDL) and BDL + AT1-R antagonist, irbesartan. Real time RT-PCR was utilised to assess gene expression of the AT1 receptor, TGF-beta1 and alpha1 (I) collagen in the liver. TGF-beta1 and alpha1 (I) collagen mRNA expression and localisation were also assessed by in situ hybridisation. TGF-beta1 activity was assessed by using the TGF-beta inducible gene product betaig-h3. Fibrosis was assessed by the Knodell scoring system, tissue hydroxyproline content and picro-sirius red staining. RESULTS Real time RT-PCR revealed that there was a 6-fold up-regulation in AT1 receptor expression in BDL animals compared with shams. This was associated with marked increases in TGF-beta1, betaig-h3 and alpha1 (I) collagen gene expression which were attenuated by AT1-RA treatment. However, AT1-RA therapy produced no significant change in liver histology or hydroxyproline content. CONCLUSIONS These results suggest that in the liver angiotensin II may play an important role in the fibrogenic response to injury. However, whether treatment with an AT1-RA will be of therapeutic benefit remains to be determined.

GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction.

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.

Cadherins in the human placenta – epithelial–mesenchymal transition (EMT) and placental development

Colonisation of the maternal uterine wall by the trophoblast involves a series of alterations in the behaviour and morphology of trophoblast cells. Villous cytotrophoblast cells change from a well-organised coherently layered phenotype to one that is extravillous, acquiring a proliferative, migratory and invasive capacity, to facilitate fetal-maternal interaction. These changes are similar to those of other developmental processes falling under the umbrella of an epithelial-mesenchymal transition (EMT). Modulation of cell adhesion and cell polarity occurs through changes in cell-cell junctional molecules, such as the cadherins. The cadherins, particularly the classical cadherins (e.g. Epithelial-(E)-cadherin), and their link to adaptors called catenins at cell-cell contacts, are important for maintaining cell attachment and the layered phenotype of the villous cytotrophoblast. In contrast, reduced expression and re-organization of cadherins from these cell junctional regions promote a loosened connection between cells, coupled with reduced apico-basal polarity. Certain non-classical cadherins play an active role in cell migration processes. In addition to the classical cadherins, two other cadherins which have been reported in placental tissues are vascular endothelial (VE) cadherin and cadherin-11. Cadherin molecules are well placed to be key regulators of trophoblast cell behaviour, analogous to their role in other developmental EMTs. This review addresses cadherin expression and function in normal and diseased human placental tissues, especially in fetal growth restriction and pre-eclampsia where trophoblast invasion is reduced.

The ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction

The efflux pump ATP binding cassette superfamily member G2 (ABCG2)/breast cancer resistance protein (BCRP) is highly expressed in human placenta. We have investigated the role of BCRP in the protection of the human placental trophoblasts from apoptosis and its expression in idiopathic fetal growth restriction, a condition associated with abnormal pla‐cental apoptosis. Inhibition of BCRP activity with the selective inhibitor Ko143 augmented cytokine (tumor necrosis factor‐α/interferon‐γ)‐induced apoptosis and phosphatidylserine externalization in primary tropho‐blast and trophoblast‐like BeWo cells. Silencing of BCRP expression in BeWo cells significantly increased their sensitivity to apoptotic injury in response to cytokines and exogenous C6 and C8 ceramides. BCRP silencing also increased intracellular ceramide levels after cytokine exposure but did not affect cellular protoporphyrin IX concentrations or sensitivity to activators of the intrinsic apoptotic pathway. BCRP expression in placentas from pregnancies complicated by idiopathic fetal growth restriction was decreased compared with controls, suggesting reduced transport of its substrates from the placenta. We conclude that BCRP may play a hitherto unrecognized survival role in the placenta, protecting the trophoblast against cytokine‐induced apoptosis and possibly other extrinsic activators via modulation of ceramide signaling. Decreased placental BCRP expression may result in reduced viability and hence functional deficit, contributing to the fetal growth restriction phenotype.—Evseenko, D. A., Murthi, P., Paxton, J. W., Reid, G., Emerald, B. S., Mohankumar, K. M., Lobie, P. E., Brennecke, S. P., Kalionis, B. Keelan, J. A. The ABC transporter BCRP/ ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction. FASEB J. 21, 3592–3605 (2007)

Changes in culture expanded human amniotic epithelial cells: implications for potential therapeutic applications.

Human amniotic epithelial cells (hAEC) isolated from term placenta have stem cell-like properties, differentiate into tissue specific cells and reduce lung and liver inflammation and fibrosis following transplantation into disease models established in mice. These features together with their low immunogenicity and immunosuppressive properties make hAEC an attractive source of cells for potential therapeutic applications. However, generation of large cell numbers required for therapies through serial expansion in xenobiotic-free media may be a limiting factor. We investigated if hAEC could be expanded in xenobiotic-free media and if expansion altered their differentiation capacity, immunophenotype, immunosuppressive properties and production of immunomodulatory factors. Serial expansion in xenobiotic-free media was limited with cumulative cell numbers and population doubling times significantly lower than controls maintained in fetal calf serum. The epithelial morphology of primary hAEC changed into mesenchymal-stromal like cells by passage 4–5 (P4–P5) with down regulation of epithelial markers CK7, CD49f, EpCAM and E-cadherin and elevation of mesenchymal-stromal markers CD44, CD105, CD146 and vimentin. The P5 hAEC expanded in xenobiotic-free medium differentiated into osteocyte and alveolar epithelium-like cells, but not chondrocyte, hepatocyte, α- and β-pancreatic-like cells. Expression of HLA Class IA, Class II and co-stimulatory molecules CD80, CD86 and CD40 remained unaltered. The P5 hAEC suppressed mitogen stimulated T cell proliferation, but were less suppressive compared with primary hAEC at higher splenocyte ratios. Primary and P5 hAEC did not secrete the immunosuppressive factors IL-10 and HGF, whereas TGF-β1 and HLA-G were reduced and IL-6 elevated in P5 hAEC. These findings suggest that primary and expanded hAEC may be suitable for different cellular therapeutic applications.

Anti-inflammatory effects of adult stem cells in sustained lung injury: a comparative study.

Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-β following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury.

Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl4) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2×106) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis.

Novel Homeobox Genes are Differentially Expressed in Placental Microvascular Endothelial Cells Compared with Macrovascular Cells

Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the microvasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. We have recently shown that placental homeobox genes HLX, DLX3, DLX4, MSX2 and GAX are expressed in placental endothelial cells. Hence, the novel homeobox genes TLX1, TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were detected that have not been reported in endothelial cells previously. Importantly, these homeobox genes have not been previously reported in placental endothelial cells and, with the exception of HEX, PHOX1 and HOXB7, have not been described in any other endothelial cell type. Reverse transcriptase PCR was performed on cDNA from freshly isolated placental microvascular endothelial cells (PLEC), and the human placental microvascular endothelial cell line HPEC. cDNAs prepared from control term placentae, human microvascular endothelial cells (HMVEC) and human umbilical vein macrovascular endothelial cells (HUVEC) were used as controls. PCR analyses showed that all novel homeobox genes tested were expressed by all endothelial cells types. Furthermore, real-time PCR analyses revealed that homeobox genes TLX1, TLX2 and PHOX1 relative mRNA expression levels were significantly decreased in HUVEC compared with microvascular endothelial cells, while the relative mRNA expression levels of MEIS2 and TGIF were significantly increased in macrovascular cells compared with microvascular endothelial cells. Thus we have identified novel homeobox genes in microvascular endothelial cells and have shown that homeobox genes are differentially expressed between micro- and macrovascular endothelial cells.

Decidua Parietalis-Derived Mesenchymal Stromal Cells Reside in a Vascular Niche Within the Choriodecidua

Mesenchymal stromal cells (MSCs) from gestational tissues represent promising cell populations with stem cell-like properties for use in regenerative medicine. Previously, we reported that MSCs in the chorionic villi of the human placenta reside in a vascular niche. However, the niche(s) in which MSCs reside in the fetal membranes, another rich source of MSCs, remains to be determined. The cell surface markers STRO-1 and 3G5 were previously employed to identify niches in a variety of tissues and here we use these markers to report the location of the MSC niche in the human decidua parietalis. The cultured decidua parietalis MSCs (DPMSCs) isolated from the choriodecidua component of the fetal membranes possessed stem cell-like properties such as adherence to plastic, colony forming ability, and multipotent differentiation potential. Fluorescence in situ hybridization analysis showed cultured DPMSCs were of maternal origin. Immunocytochemistry demonstrated that cultured DPMSCs stained positively with stem cell surface markers 3G5, CD105, CD106, STRO-1, CD146, CD49a, and α-SMA but were negative for hematopoietic markers (CD117, CD34) and vascular markers (CD34, von Willebrand factor [vWF]). Immunohistochemistry with antibodies to stem cell surface markers and the endothelial markers on term fetal membranes revealed a vascular niche for DPMSCs, which was confirmed by immunofluorescence analysis. Both STRO-1 and vWF fluorescence signals showed substantial overlap, while CD146 and vWF signals showed partial overlap. These observations were consistent with a vascular niche.

Early screening for preeclampsia

Preeclampsia, which affects about 3 to 5% of pregnant women, is the most frequent medical complication in pregnancy and the most important cause of maternal and perinatal morbidity and mortality. During the past three decades, numerous clinical, biophysical, and biochemical screening tests have been proposed for the early detection of preeclampsia. Literature shows large discrepancies in the sensitivity and predictive value of several of these tests. No single screening test used for preeclampsia prediction has gained widespread acceptance into clinical practice. Instead, its value seems to be in increasing the predictive value of panels of tests, which include other clinical measurements. The aim of this review was to examine the combination of maternal risk factors, mean arterial blood pressure, and uterine artery Doppler, together with biomarkers in the preeclampsia prediction.