Integrated molecular analysis identifies a conserved pericyte gene signature in zebrafish.

Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, and kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.

PDGFRß plays an essential role in patient vitreous-stimulated contraction of retinal pigment epithelial cells from epiretinal membranes.

Platelet-derived growth factor (PDGF) is associated with clinical proliferative vitreoretinopathy (PVR), which is characterized by formation of sub- or epi-retinal membranes that consist of cells including retinal pigment epithelial （RPE） cells and extracellular matrix. RPE cells play an important role in PVR pathogenesis. Previous findings indicated that PDGF receptor (PDGFR)α was essential in experimental PVR induced by fibroblasts. In RPE cells derived from epiretinal membranes from patients with PVR (RPEMs)， Akt was activated by PDGF-B but not PDGF-A, which suggested that PDGFRß was the predominant PDGFR isoform expressed in RPEMs. Indeed, CRISPR/Cas9-mediated depletion of PDGFRß in RPEMs attenuated patient vitreous-induced Akt activation and cellular responses intrinsic to PVR including cell proliferation, migration, and contraction. We conclude that PDGFRß appears to be the PVR relevant PDGFR isoform in RPEMs.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-ß signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-ß (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-ß results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-ß becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-ß signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a 'drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK, p44/42 MAPK and PDGFRß.

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor ß (PDGFRß) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRß phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRß phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.

Differential expression of PDGFRB and EGFR in microvascular proliferation in glioblastoma.

Glioblastoma (GBM) is the highly malignant glioma and exhibits microvascular proliferation. PCR mRNA arrays and immunohistochemical stains on tissue microarray demonstrated that the expression level of PDGFRB in GBM microvascular proliferation was significantly higher than that in GBM tumor cells while the expression level of EGFR was lower in microvascular proliferation than in GBM tumor cells. PDGFRB protein was selectively expressed in pericytes in GBM microvascular proliferation. By analyzing The Cancer Genome Atlas (TCGA) datasets for GBM, it was found that genomic DNA alterations were the main reason for the high expression of EGFR in GBM tumor cells. Our miRNA microarray data showed that microRNAs (miRNAs) (miR-193b-3p, miR-518b, miR-520f-3p, and miR-506-5p) targeting PDGFRB were downregulated in microvascular proliferation, which might be the most likely reason for the high expression of PDGFRB in GBM microvascular proliferation. The increase of several miRNAs (miR-133b, miR-30b-3p, miR-145-5p, and miR-146a-5p) targeting EGFR in GBM microvascular proliferation was one of the reasons for the lack of expression of EGFR in GBM microvascular proliferation. These findings implicated that miRNAs, such as miR-506, miR-133b, miR-145, and miR-146a, that target PDGFRB or EGFR, might be potential therapeutic agents for GBM. A new generation of targeted therapeutic agents against both EGFR and PDGFRB might be developed in the future.

Transplantation of EPCs overexpressing PDGFR-ß promotes vascular repair in the early phase after vascular injury.

BACKGROUND: Endothelial progenitor cells (EPCs) play important roles in the regeneration of the vascular endothelial cells (ECs). Platelet-derived growth factor receptor (PDGFR)-ß is known to contribute to proliferation, migration, and angiogenesis of EPCs, this study aims to investigate effects of transplantation of EPCs overexpressing PDGFR-ß on vascular regeneration. METHODS: We transplanted genetically modified EPCs overexpressing PDGFR-ß into a mouse model with carotid artery injury. After 3 days of EPCs transplantation, the enhanced green fluorescent protein (EGFP)-expressing cells were found at the injury site and the lining of the lumen by laser scanning confocal microscope (LSCM). At 4, 7, and 14 days of the carotid artery injury, reendothelialization was evaluated by Evans Blue staining. Neointima formation was evaluated at day 14 with hematoxylin and eosin (HE) staining by calculating the neointimal area, medial area, and neointimal/media (NI/M) ratio. Intimal cell apoptosis was evaluated using TUNEL assay. Then we tested whether PDGF-BB-induced VSMC migration and PDGF-BB's function in reducing VSMC apoptosis can be attenuated by EPCs overexpressing PDGFR-ß in a transwell co-culture system. RESULTS: Our results showed that EPCs overexpressing PDGFR-ß accelerates reendothelialization and mitigates neointimal formation at 14 days after injury. Moreover, we found that there is great possibility that EPCs overexpressing PDGFR-ß enhanc VSMC apoptosis and suppress VSMC migration by competitive consumption of PDGF-BB in the early phase after carotid artery injury in mice. CONCLUSIONS: We report the first in vivo and in vitro evidence that transplantation of genetically modified EPC can have a combined effect of both amplifying the reendothelialization capacity of EPCs and inhibiting neointima formation so as to facilitate better inhibition of adverse remodeling after vascular injury.

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRß (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRß RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRß-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRß or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRß or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Cited2 is required in trophoblasts for correct placental capillary patterning.

CITED2 is a transcriptional co-factor with important roles in many organs of the developing mammalian embryo. Complete deletion of this gene causes severe malformation of the placenta, and results in significantly reduced embryonic growth and death from E14.5. The placenta is a complex organ originating from cells derived from three lineages: the maternal decidua, the trophectoderm, and the extra-embryonic mesoderm. Cited2 is expressed in many of these cell types, but its exact role in the formation of the placenta is unknown. Here we use a conditional deletion approach to remove Cited2 from overlapping subsets of trophectoderm and extra-embryonic mesoderm. We find that Cited2 in sinusoidal trophoblast giant cells and syncytiotrophoblasts is likely to have a non-cell autonomous role in patterning of the pericytes associated with the embryonic capillaries. This function is likely to be mediated by PDGF signaling. Furthermore, we also identify that loss of Cited2 in syncytiotrophoblasts results in the subcellular mislocalization of one of the major lactate transporters in the placenta, SLC16A3 (MCT4). We hypothesize that the embryonic growth retardation observed in Cited2 null embryos is due in part to a disorganized embryonic capillary network, and in part due to abnormalities of the nutrient transport functions of the feto-maternal interface.

ß-PDGF receptor expressed by hepatic stellate cells regulates fibrosis in murine liver injury, but not carcinogenesis.

BACKGROUND & AIMS: Rapid induction of ß-PDGF receptor (ß-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of ß-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent ß-PDGFR activity, and assessed its prognostic implications in human cirrhosis. METHODS: The impact of either loss or constitutive activation of ß-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked ß-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis. RESULTS: Depletion of ß-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-κB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of ß-PDGFR in stellate cells. In the human cohort, the ß-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis. CONCLUSIONS: ß-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Platelet-derived growth factor receptor ß (PDGFRß) immunohistochemistry highlights activated bone marrow stroma and is potentially predictive for fibrosis progression in prefibrotic myeloproliferative neoplasia.

AIMS: Myelofibrosis is the result of aberrant stromal activity which is determined routinely by reticulin staining in bone marrow biopsies. As matrix fibres are the product of activated fibroblasts, we analysed fibre accumulation compared to stromal cell activity during myelofibrosis progression using the fibroblast activation marker platelet-derived growth factor receptor ß (PDGFRß) by immunohistochemistry. METHODS AND RESULTS: Initial and follow-up bone marrow biopsies from 84 patients with myeloproliferative neoplasia, including 55 cases with primary myelofibrosis, were evaluated from five haematopathology centres. The stromal mass was measured by conventional reticulin staining [myelofibrosis (MF) grade, 0-3] and PDGFRß-positive cells using a novel PDGFRß scoring system (0-3). Results were correlated for prediction of progression. The MF grade and the PDGFRß score showed excellent correlation (Spearman's r = 0.83, P < 0.0001). Elevated PDGFRß scores (higher than MF-grade) predicted myelofibrosis progression in total with 43% sensitivity and 57% specificity, and short-term (within 1 year) progression with 82% sensitivity and 53% specificity. Progression of prefibrotic disease to manifest myelofibrosis could be forecast with 90% sensitivity and 75% specificity. CONCLUSION: PDGFRß highlights stromal cell activation in marrow fibrosis, which is closely related to matrix accumulation, indicating a direct clinical impact especially in prefibrotic myeloproliferative disorders.

Levels of active tyrosine kinase receptor determine the tumor response to Zalypsis.

BACKGROUND: Zalypsis(®) is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing's sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy. METHODS: Using in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies. RESULTS: Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/ß, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug. CONCLUSIONS: RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials.

Platelet-derived growth factor receptor-ß expression in human peritoneum.

INTRODUCTION: Simple peritoneal fibrosis and encapsulating peritoneal sclerosis (EPS) are important lesions in the peritoneum of patients on peritoneal dialysis (PD). We have previously described a population of podoplanin-positive myofibroblasts in peritoneal biopsies from patients with EPS. Platelet-derived growth factor receptor-ß (PDGFRß) is a marker of pericytes, and PDGFs might be involved in the fibrotic response of the peritoneum. This study aimed to describe PDGFRß in the human peritoneum. METHODS: In this retrospective analysis, we localized PDGFRß in peritoneal biopsies from patients with EPS (n = 6) and patients on PD without signs of EPS (n = 5), and compared them with normal peritoneum (n = 4) and peritoneum from uremic patients (n = 5). Consecutive sections were stained for smooth-muscle actin (SMA) and podoplanin. Slides were scored semiquantitatively by 2 observers blinded to the diagnosis. RESULTS: PDGFRß was expressed by cells of arterial walls in all biopsies. A prominent population of PDGFRß-positive cells was present in the normal peritoneum, which were SMA negative on consecutive sections. In patients on PD, a high number of PDGFRß were also positive for SMA. In EPS, the majority of podoplanin-positive cells were positive for PDGFRß. In peritoneal biopsies from normal and uremic patients, the expression of SMA was mainly restricted to cells of arterial walls. Podoplanin expression was restricted to lymphatic vessels in normal peritoneum, in uremic patients, and in patients on PD without EPS. CONCLUSIONS: As podoplanin-positive myofibroblasts express PDGFRß, these cells might be related to pericytes (rather than other sources of fibroblasts). PDGFRß might turn out to be a therapeutic target in EPS.

Renal ischemia-reperfusion induces a dysbalance of angiopoietins, accompanied by proliferation of pericytes and fibrosis.

Endothelial cells (ECs) are highly susceptible to hypoxia and easily affected upon ischemia-reperfusion (I/R) during renal transplantation. Pericytes and angiopoeitins play important role in modulating EC function. In the present study, we investigate the effect of renal I/R on the dynamics of angiopoietin expression and its association with pericytes and fibrosis development. Male Lewis rats were subjected to unilateral renal ischemia for 45 min followed by removal of the contralateral kidney. Rats were killed at different time points after reperfusion. Endothelial integrity (RECA-1), pericytes [platelet-derived growth factor receptor-ß (PDGFR-ß)], angiopoietin-2 (Ang-2)/angiopoietin-1 (Ang-1) expression, and interstitial collagen deposition (Sirius red and α-smooth muscle actin) were assessed using immunohistochemistry and RT-PCR. Our study shows an increase in protein expression of Ang-2 starting at 5 h and remaining elevated up to 72 h, with a consequently higher Ang-2/Ang-1 ratio after renal I/R (P < 0.05 at 48 h). This was accompanied by an increase in protein expression of the pericytic marker PDGFR-ß and a loss of ECs (both at 72 h after I/R, P < 0.05). Nine weeks after I/R, when renal function was restored, we observed normalization of the Ang-2/Ang-1 ratio and PDGFR-ß expression and increase in cortical ECs, which was accompanied by fibrosis. Renal I/R induces a dysbalance of Ang-2/Ang-1 accompanied by proliferation of pericytes, EC loss, and development of fibrosis. The Ang-2/Ang-1 balance was reversed to baseline at 9 wk after renal I/R, which coincided with restoration of cortical ECs and pericytes. Our findings suggest that angiopoietins and pericytes play an important role in renal microvascular remodeling and development of fibrosis.

Alteration of circulating miRNAs in SSc: miR-30b regulates the expression of PDGF receptor ß.

OBJECTIVE: Although several miRNAs have been shown to regulate autoimmune pathogenesis by affecting lymphocyte function, the roles of miRNAs in the pathogenesis of SSc remain unclear. Therefore the purpose of this study was to identify miRNAs that play a role in the pathogenesis of SSc by quantitative PCR screening of serum miRNAs. METHODS: Ninety-five miRNAs that were predicted to target SSc-related genes [IL-4, TGF-ß, CTGF, PDGF-B, PDGF receptor (PDGFR) α/ß and COL1A2) by in silico analyses were selected. The expression of these miRNAs in sera of SSc patients and healthy controls was measured by quantitative PCR. Involvement of miR-30b, which was most strongly down-regulated in SSc patients, in the regulation of PDGFR-ß expression was examined by transfection experiments and 3'-untranslated region (3'-UTR) target luciferase assays. The expression of miR-30b in skin was evaluated in a bleomycin-induced dermal fibrosis model in mice and in SSc patients. RESULTS: Nineteen of 95 miRNAs were significantly decreased in the sera of SSc patients. Among them, miR-30b was most strongly down-regulated in SSc patients (P = 0.00006) and the levels of miR-30b were inversely correlated with modified Rodnan skin scores. Transfection of a miR-30b mimic repressed PDGFR-ß expression in dermal fibroblasts and the activity of a luciferase reporter containing 3'-UTR of PDGFR-ß. Moreover, the expression of miR-30b was down-regulated in bleomycin-treated sclerotic skin and in affected skin in SSc patients. CONCLUSION: Down-regulation of miR-30b might be involved in the pathogenesis of SSc.

Adventitial pericyte progenitor/mesenchymal stem cells participate in the restenotic response to arterial injury.

Restenosis is a major complication of coronary angioplasty, at least partly due to the fact that the origin and identity of contributing cell types are not well understood. In this study, we have investigated whether pericyte-like cells or mesenchymal stem cells (MSCs) from the adventitia contribute to restenosis. We demonstrate that while cells expressing the pericyte markers NG2, platelet-derived growth factor receptor ß, and CD146 are rare in the adventitia of uninjured mouse femoral arteries, following injury their numbers strongly increase. Some of these adventitial pericyte-like cells acquire a more MSC-like phenotype (CD90+ and CD29+ are up-regulated) and also appear in the restenotic neointima. Via bone marrow transplantation and ex vivo artery culture approaches, we demonstrate that the pericyte-like MSCs of the injured femoral artery are not derived from the bone marrow, but originate in the adventitia itself mainly via the proliferation of resident pericyte-like cells. In summary, we have identified a population of resident adventitial pericyte-like cells or MSCs that contribute to restenosis following arterial injury. These cells are different from myofibroblasts, smooth muscle cells, and other progenitor populations that have been shown to participate in the restenotic process.

Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-ß and VEGFR-2 expression.

Despite intensive research studies, theories have yet to focus on the contribution of hypoxia to patency differences observed clinically between arterial vs. venous grafts. This study investigates the differential hypoxic response of smooth muscle cells (SMC) to hypoxia-derived endothelial cell (EC) growth factors. Initiation of SMC proliferation under hypoxia (<5% O(2)) occurred only after incubation with hypoxic endothelial cell-conditioned media (H-ECM). After the investigation of several possible growth factors in the H-ECM that may be responsible for SMC proliferation, the greatest difference was observed in vascular endothelial growth factor (VEGF-A) and platelet-derived growth factor homodimer B (PDGF-BB) expression. VEGF-A increased (2-fold) significantly (P < 0.05) in arterial-derived smooth muscle cells (ASMC) under hypoxia compared with venous-derived smooth muscle cells (VSMC), which showed no significant change. VSMC showed significant (P < 0.05) increase in VEGFR-2 expression under hypoxia compared with ASMC. Incubation with VEGFR-2-neutralizing antibody/PDGFR antagonist in VSMC before addition of H-ECM resulted in decreased proliferation. ASMC proliferation under hypoxia did not decrease during incubation with VEGFR-2-neutralizing antibody but did decrease upon PDGFR antagonist incubation. Current therapies focusing on treating intimal hyperplasia have negated the fact that combinational therapy might be required to combat induction of SMC proliferation. Clinically, therapy with PDGFR antagonists plus anti-VEGFR-2 may prove to be efficacious in managing SMC proliferation in venous-derived grafts.

Establishment of conditionally immortalized human glomerular mesangial cells in culture, with unique migratory properties.

The aim of this study was to establish an immortalized human mesangial cell line similar to mesangial cells in vivo for use as a tool for understanding glomerular cell function. Mesangial cells were isolated from glomerular outgrowths from a normal human kidney, then retrovirally transfected with a temperature-sensitive SV40T antigen+human telomerase (hTERT). Mesangial cells exhibited features of compact cells with small bodies in a confluent monolayer at 33°C, but the cell shape changed to flat and stellate after 5 days in growth-restrictive conditions (37°C). Western blot and immunofluorescence analysis showed that podocyte markers (nephrin, CD2AP, podocin, Wilms' tumor-1) and an endothelial-specific molecule (VE-cadherin) were not detectable in this cell line, whereas markers characteristic of mesangial cells (α-SMA, fibronectin, and PDGFß-R) were strongly expressed. In migration assays, a significant reduction in wound surface was observed in podocyte and endothelial cells as soon as 12 h (75 and 62%, respectively) and complete wound closure after 24 h. In contrast, no significant change was observed in mesangial cells after 12 h, and even after 48 h the wounds were not completely closed. Until now, conditionally immortalized podocyte and endothelial cell lines derived from mice and humans have been described, and this has greatly boosted research on glomerular physiology and pathology. We have established the first conditionally immortalized human glomerular mesangial cell line, which will be an important adjunct in studies of representative glomerular cells, as well as in coculture studies. Unexpectedly, mesangial cells' ability to migrate seems to be slower than for other glomerular cells, suggesting this line will demonstrate functional properties distinct from previously available mesangial cell cultures. This conditionally immortalized human mesangial cell line represents a new tool for the study of human mesangial cell biology in vitro.

Multipotent PDGFRß-expressing cells in the circulation of stroke patients.

Tissue pericytes respond to injury, and support vascular and tissue regeneration. The presence of pericytes in the circulation may provide an attractive framework for tissue regeneration. Here, we detected multipotent pericyte-like cells in the circulating blood and determined its profiles during cerebral ischemia. Pericyte-like cells were isolated from the peripheral blood of acute stroke patients or asymptomatic individuals with vascular risk factors by fluorescence or magnetic activated cell sorting with anti-PDGF receptor-beta (PDGFRß) antibody. The morphologic and molecular features of circulating PDGFRß(+) cells were compared with tissue pericytes, and the associations with respect to quantity in the blood, culture outcome, and patient characteristics were analyzed. We found an increase in circulating PDGFRß(+) cells in acute stroke patients compared to controls and a correlation with neurologic impairment. The isolated PDGFRß(+) cells expressed mesenchymal stem cell markers, proliferated, and were multipotent under permissive culture conditions. The multipotent nature of these cells was comparable to fat-derived PDGFRß(+) cells. These cells could be obtained by pharmacologic stimulation using bone marrow mobilizer. Circulating PDGFRß(+) cells will be useful for future research involving endogenous recovery or autologous cell-based therapy.

Hepatocyte apoptotic bodies encasing nonstructural HCV proteins amplify hepatic stellate cell activation: implications for chronic hepatitis C.

Chronic hepatitis C infection leads to increased hepatocyte apoptosis. Because engulfment of apoptotic bodies (ABs) by hepatic stellate cells (HSC) is profibrogenic, we compared the effects of ABs derived from hepatitis C virus (HCV)-negative vs HCV-infected (Con1+) Huh7 hepatoblastoma cells on fibrogenic and activation-related mRNA expression by a human HSC line (LX2). Uptake of Huh7(Con1+) ABs by LX2 cells dose dependently upregulated profibrotic genes (COL1A1, TGFB1; TIMP1; TIMP2). When normalized to the apoptotic cytokeratin-18 M30 neoepitope, HCV(+) ABs exhibited a more pronounced effect than HCV(-) ABs. In contrast, neither noningested ABs nor nucleic acids obtained from Huh7, Huh7(Con1+) or HepG2 cells triggered those AB-dependent effects. Both the engulfment of Huh7(Con1+) ABs and their effects were partially blocked by masking of phosphatidylserine with annexin V and completely inhibited by the class-A scavenger receptor ligand, polyinosinic acid. Our findings demonstrate that AB uptake stimulates HSCs and indicate that HCV infection leads to amplified fibrogenic mRNA expression and enhanced HSC activation.

Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface.

BACKGROUND: Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points. METHODS: Mesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections. RESULTS: At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50. CONCLUSIONS: We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.