Kim A. Butters

Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human‐pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens.

Lumican, an extracellular matrix proteoglycan, is a novel requisite for hepatic fibrosis

Lumican, an extracellular matrix proteoglycan was previously shown to be upregulated with increasing severity of nonalcoholic steatohepatitis (NASH). Although lumican is involved in collagen fibrillogenesis in extra-hepatic tissues, little is known about the role of lumican in hepatic disease. We therefore determined lumican expression in etiologies other than clinical NASH. Our results indicated that lumican is upregulated in clinical samples of hepatitis C virus infection, in experimental rodent models of chronic and acute liver injury and could additionally be induced in vitro in response to the pro-fibrotic cytokine transforming growth factor β1 (TGFβ1) and to lipotoxic palmitic acid. Together, these results suggested a role for lumican in hepatic fibrosis. To investigate the functional role of lumican in hepatic fibrosis, lumican null (Null) and wild-type (WT) littermates were administered carbon tetrachloride intra-peritoneally. Serum and liver tissue were analyzed for indices of liver injury, fibrosis, matrix turnover, and proliferation. Hepatic fibrosis was greatly reduced in null animals (P<0.05). Paradoxically, gene expression of fibrosis-related genes such as TGFβ1 and collagen 1 was numerically higher in null animals though statistically insignificant from WT animals. On the other hand, α smooth muscle actin expression (α-SMA), a marker for activated fibroblasts, the main contributors of collagen production was significantly higher (P<0.05) in null animals as compared with WT littermates. Among the matrix metalloproteases (MMP), MMP13 was significantly increased (P<0.05) in null animals. Ultra-structural imaging indicated differences in the organization and spatial distribution of hepatic collagen fibrils of null and WT mice. Cell proliferation was significantly increased (P<0.05) in null animals. We conclude that lumican is a prerequisite for hepatic fibrosis. The protective effect of lumican deficiency in hepatic fibrosis appears to be downstream of collagen production and mediated through the combined effects of impaired collagen fibrillogenesis, increased matrix turnover, and an enhanced proliferative response.

Intrinsic resistance of hepatocytes to complement-mediated injury

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.

Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model.

Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 MAPK as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established a murine RAS model by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 wk. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation. In mice treated with SB203580, the RAS-induced renal atrophy was reduced (70% vs. 39%, P < 0.05). SB203580 also reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 partially blocked the induction of CCL2, CCL7 (MCP-3), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidneys. In vitro, blockade of p38 hindered both TNF-α and TGF-β-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.

Persistent Urinary Podocyte Loss following Preeclampsia May Reflect Subclinical Renal Injury

Objective Studies have shown that podocyturia, i.e., urinary loss of viable podocytes (glomerular epithelial cells), is associated with proteinuria in preeclampsia. We postulated that urinary podocyte loss may persist after preeclamptic pregnancies, thus resulting in renal injury. This may lead to future chronic renal injury. In addition, we compared the postpartum levels of the angiogenic factors, which previously have been associated with preeclampsia, between normotensive versus preeclamptic pregnancies. Study Design The diagnosis of preeclampsia was confirmed using standard clinical criteria. Random blood and urine samples were obtained within 24 hours prior to delivery and 5 to 8 weeks postpartum. Urine sediments were cultured for 24 hours to select for viable cells and staining for podocin was used to identify podocytes. Serum samples were analyzed for the levels of angiogenic markers using ELISA (enzyme-linked immunosorbent assay) methodology. Results At delivery, preeclamptic patients (n = 10) had significantly higher proteinuria (p = 0.006) and podocyturia (p<0.001) than normotensive pregnant patients (n = 18). Postpartum proteinuria was similar between these two groups (p = 0.37), while podocyturia was present in 3 of 10 women with preeclampsia and in none of the normotensive controls (p = 0.037). Angiogenic marker levels, including placental growth factor, soluble vascular endothelial growth factor receptor fms-like tyrosine kinase receptor-1 and endoglin, were not significantly different between women with preeclampsia and women with a normotensive pregnancy, either at delivery or postpartum. Conclusion Persistent urinary podocyte loss after preeclamptic pregnancies may constitute a marker of ongoing, subclinical renal injury.

Toward the survival and function of xenogeneic hepatocyte grafts

Xenogeneic hepatocyte transplantation might offer an unobtrusive alternative to whole liver allotransplantation. Having previously found that the immune response to such grafts can be controlled by immunosuppression, we sought approaches to collection and delivery that would optimize survival and function after transplantation. Porcine hepatocytes were isolated by a 2‐step collagenase technique and then: 1) used immediately; 2) stored in University of Wisconsin (UW) solution at 4°C; 3) cultured in supplemented Williams E medium; or 4) cryopreserved in UW solution with 10% dimethyl sulfoxide (DMSO). The fate and function of the hepatocytes was determined after they were injected into the spleens of immunodeficient mice. Freshly isolated hepatocytes had better viability (92.2 ± 1.9%) than hepatocytes cultured for 24 hours (78.4 ± 6.3%), hypothermically preserved in UW solution for 24 hours (85.8 ± 3.1%), or cryopreserved (65.0 ± 2.6%). Freshly isolated hepatocytes secreted more albumin after transplantation than hepatocytes that were cultured, hypothermically stored, or cryopreserved. In conclusion, culture and storage profoundly compromises the function of isolated hepatocytes after transplantation. Freshly isolated hepatocytes are the preferred source for transplantation. (Liver Transpl 2005;11:39–50.)

Molecular bioluminescence imaging as a noninvasive tool for monitoring tumor growth and therapeutic response to MRI-guided laser ablation in a rat model of hepatocellular carcinoma

ObjectivesThe objective of this study was to quantitatively compare tumor imaging by magnetic resonance imaging (MRI) and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2-dimensional BLI and 3-dimensional diffuse luminescence tomography (3D DLIT) in an orthotopic rat model of hepatocellular carcinoma. Materials and MethodsThis study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (n = 3) or a heat shock element luciferase reporter (HSE-luc; n = 4) into the liver. All rats underwent MRI to assess tumor establishment and volume and 2-dimensional BLI to assess tumor luminescence at day 7 with subsequent MRI and 2D BLI and 3D DLIT in select animals at days 14 and 21. Magnetic resonance imaging–guided laser ablation of the tumor was performed with preablation and postablation 2D BLI and/or 3D DLIT (n = 2). The tumors underwent histopathologic analysis to assess tumor viability. ResultsThe MRI scans demonstrated hyperintense T2-weighted lesions at 3 of 3 and 4 of 4 sites in the empty vector and HSE-luc rats, respectively. Two-dimensional BLI quantitation demonstrated 23.0-fold higher radiance in the HSE-luc group compared with the empty vector group at day 7 (P < 0.01) and a significant correlation with tumor volume by MRI (r = 0.86; P < 0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. Three-dimensional DLIT quantitation demonstrated better agreement with thepercentage of nonviable tumor by histopathology than did 2D BLI quantitation after the MRI-guided laser ablation. ConclusionsBioluminescence imaging is feasible as a noninvasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of hepatocellular carcinoma.

In utero cell transfer between porcine littermates

Trafficking of cells between mother and fetus during the course of normal pregnancy is well documented. Similarly, cells are known to travel between twins that share either a placenta (i.e. monozygotic) or associated chorion (i.e. monochorionic). Transferred cells are thought to be channelled via the vessels of the placenta or vascular connections established via the chorion and the long-term presence of these cells (i.e. microchimerism) can have important consequences for immune system function and reparative capacity of the host. Whether cells can be transferred between twins with separate placentas and separate chorions (i.e. no vascular connections between placentas) has not been investigated nor have the biological consequences of such a transfer. In the present study, we tested the possibility of this type of cell transfer by injecting human cord blood-derived cells into a portion of the littermates of swine and probing for human cells in the blood and tissues of unmanipulated littermates. Human cells were detected in the blood of 78% of unmanipulated littermates. Human cells were also detected in various tissues of the unmanipulated littermates, including kidney (56%), spleen (33%), thymus (11%) and heart (22%). Human cells were maintained in the blood until the piglets were sacrificed (8 months after birth), suggesting the establishment of long-term microchimerism. Our findings show that the transfer of cells between fetuses with separate placentas and separate chorions is significant and thus such twins may be subject to the same consequences of microchimerism as monozygotic or monochorionic counterparts.

Methodological differences account for inconsistencies in reported free VEGF concentrations in pregnant rats

Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/ml) and high (527-1,030 pg/ml) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Plasma VEGF in nonpregnant, day 7 pregnant, and day 19 pregnant rats was measured using rat and mouse ELISAs (R&D Systems). The rat ELISA detected VEGF in plasma from nonpregnant rats but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (median: 2,273 vs. 0 pg/ml) than in nonpregnant (97 vs. 20 pg/ml) and day 7 pregnant (66 vs. 2 pg/ml) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from nonpregnant and day 7 pregnant rats was high for the rat ELISA (82-105%) but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. The use of the rat versus mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations in nonpregnant and pregnant rats are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, these data should be interpreted cautiously.

Heat stress induced cell death mechanisms in hepatocytes and hepatocellular carcinoma: In vitro and in vivo study

The aims of the present study were to investigate the thermal‐dose dependent effect of heat stress on hepatocyte and HCC cell death mechanisms using clinically relevant experimental heat stress conditions in vitro and to investigate apoptotic cell death induced by laser thermal ablation in vivo.