Biomedical subjects

Dontscho Kerjaschki

Publications and source records attributed to Dontscho Kerjaschki.

At least 19 recordsLinked to original sources

PubMed2006

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible.

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

Animals↗

PubMed2006

Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels.

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.

Amino Acid Sequence↗

PubMed2006

Mouse endosialin, a C-type lectin-like cell surface receptor: expression during embryonic development and induction in experimental cancer neoangiogenesis.

Endosialin is a C-type lectin-like cell surface receptor of unknown function, with a distinctive pattern of endothelial expression in newly formed blood vessels in human cancers. The murine orthologue of endosialin has been identified, opening up the analysis of developmental regulation in the embryo and in aberrant tissue remodeling, notably cancer angiogenesis. To advance these studies we have generated an antibody to the extracellular domain of mouse endosialin and mapped protein expression from embryonic day E10.0 to the adult stage, complemented by mRNA quantification and co-typing for standard endothelial markers. Four main findings emerged. First, endosialin protein is restricted to vascular endothelium and fibroblast-like cells in developing organs, and largely disappears in the adult. Second, endothelial expression varies markedly between organs regarding spatial and temporal patterns. For instance, in the E10.0 embryo, endosialin is prominent in the endothelium of the dorsal aorta and, from E11.0 to E14.5, in vessels sprouting from the dorsal aorta, in perineural vascular plexuses, and in brain capillaries. Third, circumscribed mesenchymal expression in fibroblast-like cells was evident throughout development, most pronounced adjacent to certain budding epithelia, as exemplified by the lung and kidney glomeruli, but unrelated to the endothelial expression. The endosialin protein persists in the stromal fibroblasts of the adult uterus. Finally, in subcutaneous cancer xenograft models endosialin re-appears in the host-derived tumor stroma, both in neo-angiogenic vascular endothelium and in activated stromal fibroblasts. In future studies, the search for intrinsic or extrinsic signals contributing to endosialin induction in cancer stroma will be of interest.

Animals↗

PubMed2006

Everolimus inhibits glomerular endothelial cell proliferation and VEGF, but not long-term recovery in experimental thrombotic microangiopathy.

BACKGROUND: Everolimus is a potent immunosuppressant used in renal transplant therapy, but its effects on renal endothelial cell regeneration after injury are unknown. The effects of an everolimus therapy were investigated in a model of renal thrombotic microangiopathy (TMA) with specific endothelial cell (EC) injury in the rat in vivo as well as in glomerular ECs in vitro. METHODS: During the early regenerative phase (day 3) of the renal microvascular injury model in vivo, everolimus inhibited glomerular EC proliferation by up to 60% compared with vehicle-treated rats, whereas apoptosis was not different in these groups. This decreased EC proliferation was associated with an enhanced deposition of fibrin in everolimus treated animals on day 3. In cultured glomerular endothelial cells, everolimus effectively and dose dependently inhibited cellular proliferation. This anti-proliferative effect was associated with a reduced phosphorylation of the p70S6 kinase and reduction of the pro-angiogenic factor VEGF in glomeruli in vivo and in cultured podocytes in vitro. RESULTS: Despite the prolonged EC repair and in contrast to the anti-Thy1 nephritis model, everolimus therapy did not disturb the long-term repair reaction in this thrombotic microangiopathy model. CONCLUSION: Everolimus is anti-proliferative for glomerular EC in vitro and in vivo and does not seem to have detrimental long-term effects in experimental renal TMA, when only the glomerular endothelium, but not the mesangium is severely injured.

Animals↗

PubMed2006

Early lymph vessel development from embryonic stem cells.

OBJECTIVE: The purpose of this study was to establish a model system for lymph vessel development based on directed differentiation of murine embryonic stem cells. METHODS AND RESULTS: Stem cells were aggregated to form embryoid bodies, and subsequently cultured in 3-dimensional collagen matrix for up to 18 days. Treatment with vascular endothelial growth factor (VEGF)-C and VEGF-A individually enhanced formation of lymphatic vessel structures, although combined treatment with VEGF-C and VEGF-A was most potent and gave rise to a network of LYVE-1, podoplanin, Prox1, and VEGF receptor-3 positive lymphatic vessel structures running parallel to and apparently emanating from, capillaries. In contrast, fibroblast growth factor-2, hepatocyte growth factor, or hypoxia had little or no effect on the development of the early lymphatics. Further, cells of hematopoietic origin were shown to express lymphatic markers. In summary, different subpopulations of lymphatic endothelial cells were identified on the basis of differential expression of several lymphatic and blood vessel markers, indicating vascular heterogeneity. CONCLUSIONS: We conclude that the present model closely mimics the early steps of lymph vessel development in mouse embryos.

Animals↗

PubMed2006

Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants.

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.

Base Sequence↗

PubMed2006

Lymphangiogenesis in the bone-implant interface of orthopedic implants: importance and consequence.

The lymphatic system plays an essential physiological role in homeostasis, interstitial fluid composition, and immunity while impaired lymphatic function has been implicated in a number of pathological conditions, including arthritis and delayed wound healing. This study investigated lymphatic capillary growth and lymphangiogenesis pathways in the bone-implant interface from patients with aseptically loosened prosthetic joints. The newly developed lymphatic specific marker, podoplanin, has enabled the first demonstration of lymphatic capillaries in peri-prosthetic tissues (60% of cases contained podoplanin positive vessels). The pro-lymphangiogenic factor (VEGF-C) and its receptor VEGFR-3 showed high level of expression in these tissues, (often in areas of high levels of wear debris). However despite the upregulation of the lymphangiogenesis pathway by a VEGF-C/VEGFR-3-mediated mechanism, there were relatively few podoplanin positive lymphatic vessels in the bone-implant interface (3.4% of total vessels). This may have important pathological consequences in terms of perpetuating inflammation and edema by inhibiting the removal of macromolecules, cells, and interstitial fluid. The identification of lymphatic vessels with internalized polyethylene wear particles provides evidence of this route of wear debris transportation to distal sites. This paper highlights the importance of lymphatic vessels in the maintenance of local and distal inflammatory responses to prosthetic wear particles.

Aged↗

PubMed2006

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Animals↗

PubMed2006

Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton.

The expression of podoplanin, a small mucin-like protein, is upregulated in the invasive front of a number of human carcinomas. We have investigated podoplanin function in cultured human breast cancer cells, in a mouse model of pancreatic beta cell carcinogenesis, and in human cancer biopsies. Our results indicate that podoplanin promotes tumor cell invasion in vitro and in vivo. Notably, the expression and subcellular localization of epithelial markers are unaltered, and mesenchymal markers are not induced in invasive podoplanin-expressing tumor cells. Rather, podoplanin induces collective cell migration by filopodia formation via the downregulation of the activities of small Rho family GTPases. In conclusion, podoplanin induces an alternative pathway of tumor cell invasion in the absence of epithelial-mesenchymal transition (EMT).

Actins↗

PubMed2006

Laser capture microdissection of epithelial cancers guided by antibodies against fibroblast activation protein and endosialin.

Transcriptional profiling of cancer biopsies is used extensively to identify expression signatures for specific cancer types, diagnostic and prognostic subgroups, and novel molecular targets for therapy. To broaden these applications, several challenges remain. For example, the integrity of RNA extracted even from small tissue samples has to be insured and monitored. Moreover, total tumor RNA may hide the marked histologic heterogeneity of human cancers. A principle approach to this heterogeneity has been provided by laser capture microdissection performed on antibody-stained tissue sections (immuno-LCM; iLCM). In this study, we have established a procedure to assess the quality of RNA obtained from tissue sections, coupled with immunostaining using antibodies to different tumor stromal markers, and subsequent iLCM to selectively capture the cancer stroma compartments. The procedure was applied to 53 frozen specimens of human epithelial cancers. Sections were stained for histopathological evaluation, and RNA was isolated from adjacent serial sections. RNA quality was assessed by the Agilent-Bioanalyzer (Agilent, Palo Alto, CA) and by multiplex RT-PCR. Two thirds of the specimens were found to yield good to excellent RNA quality. For microdissection of the tumor stroma with reactive fibroblasts and tumor blood vessels, a rapid incubation protocol with antibodies against fibroblast activation protein (FAP) and against endosialin was developed to ensure RNA integrity for subsequent iLCM. Using these procedures, RNA from distinct tumor compartments can be isolated, analyzed, amplified, and used for transcription profiling.

Antibodies, Neoplasm↗

PubMed2006

The sialomucin CD34 is a marker of lymphatic endothelial cells in human tumors.

The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified. Applying double-staining techniques with established LEC markers, we have screened endothelial cell differentiation antigens for their expression in LECs. These experiments identified the sialomucin CD34 as being exclusively expressed by LECs in human tumors but not in corresponding normal tissues. CD34 is expressed by LYVE-1(+)/podoplanin(+)/Prox1(+) tumor-associated LECs in colon, breast, lung, and skin tumors. More than 60% of analyzed tumors contained detectable intratumoral lymphatics. Of these, more than 80% showed complete co-localization of CD34 with LEC markers. In contrast, LECs in all analyzed normal organs did not express CD34. Corresponding analyses of experimental tumors revealed that mouse tumor-associated LECs do not express CD34. Taken together, these experiments identify CD34 as the first differentially expressed LEC antigen that is selectively expressed by tumor-associated LECs. The data warrant further exploration of CD34 in tumor-associated LECs as a prognostic tumor marker.

Antigens, CD34↗

PubMed2005

Glomerular permeability is altered by loss of P0, a myelin protein expressed in glomerular epithelial cells.

The myelin protein 0 (MPZ or P0) is a transmembrane glycoprotein that represents the most abundant myelin component. Mutations in the P0 gene are associated with one form of autosomal dominant demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 1B (CMT1B). Because CMT1 may be associated with renal involvement, mostly focal segmental glomerulosclerosis, we hypothesized that P0 could be expressed in the kidney. P0 mRNA was detected by reverse transcriptase-PCR in the human and mouse renal cortex. P0 transcripts were identified by in situ hybridization at different stages of the mouse kidney development, especially in embryonic structures that give rise to the glomerulus. P0 protein was also detected by Western blot in human and rat glomerular extracts and in a human podocyte cell line using a monoclonal anti-P0 antibody. Immunofluorescence studies on human kidney sections showed that the podocytes were intensely labeled. Immunogold electron microscopy disclosed a predominant staining of the membranes of intracellular vesicles in podocytes. P0 was also detected in the podocyte cell membrane, including at the foot processes. P0(-/-) mice exhibited mild growth retardation and demyelinating neuropathy similar to the one observed in patients with CMT1B. They also presented mild albuminuria, without significant ultrastructural change of the glomerular basement membrane or the podocytes. These results demonstrate that P0, the major myelin protein, is also expressed during nephrogenesis and in mature kidney, mostly in podocytes. They suggest that P0 gene mutations might be involved in renal diseases.

Adult↗

PubMed2005

Characterization of cancer stroma markers: in silico analysis of an mRNA expression database for fibroblast activation protein and endosialin.

Standardized, high-throughput RNA detection with microarray chips allows for the construction of genome-wide databases for tissue specimens suitable for in silico electronic Northern blot (eNorthern) analysis of marker genes. We used the BioExpress database, which contains transcriptional profiles of normal and cancer samples, to examine two putative markers of cancer stroma: fibroblast activation protein-alpha (FAP-alpha) and endosialin. Analyses for FAP-alpha showed that normal tissues generally lack RNA signals, with the exception of endometrium. Typing of tumors revealed prominent FAP-alpha signals in cancer types marked by desmoplasia, and localization of FAP-alpha in reactive cancer stroma was confirmed by immunohistochemistry. A subset of sarcomas displayed prominent FAP-alpha signals localizing to the malignant cells. For endosialin, eNorthern analyses showed low to moderate RNA signals in many normal organs, whereas immunohistochemistry revealed endosialin in only some tissues, such as endometrium. Endosialin was detected at the RNA and protein level in sarcomas, notably malignant fibrous histiocytomas. Low to moderate endosialin RNA signals were found in epithelial cancer types for which immunostaining identifies expression in subsets of tumor capillaries or fibroblasts. These findings extend the FAP-alpha and endosialin profiling in silico to an unbiased tumor database and place both molecules in a novel context of endometrial biology and sarcoma subtyping. Our findings suggest that BioExpress can be searched directly for tumor stroma markers but may need prior enrichment for markers with narrow cellular representation, such as endosialin. Constructing databases from microdissected cancer tissues may be an essential step for tumor stroma-targeted therapies.

Adult↗

PubMed2005

Lymphatic spread of ductal pancreatic adenocarcinoma is independent of lymphangiogenesis.

Early lymph node metastasis is common in pancreatic ductal adenocarcinoma (PDAC). The present study has examined the relationship of lymphatic spread to lymph vessel development and the expression of lymphangiogenic cytokines in a series of well-characterized PDACs. The hot spot method revealed the intratumoural and peritumoural lymphatic vessel density (LVD) to be slightly higher in PDACs than in the normal pancreas. The average intratumoural LVD, however, was strikingly decreased. There was no overexpression of vascular endothelial growth factor (VEGF)-C and VEGF-D in PDACs compared with the normal pancreas. LVD and expression of lymphangiogenic cytokines were not related to any of the biological tumour features or to patient survival. Three orthotopic nude mouse PDAC models did not reveal any increase in tumour-associated LVD, despite a high rate of lymph node metastasis. Lymph vessel proliferation was comparable in PDAC and chronic pancreatitis, in both humans and mice. In conclusion, increased lymphangiogenic activity is not required for and does not significantly affect the lymphatic spread of PDAC. The reduced number of human and murine intratumoural lymph vessels indicates that lymphatic metastasis takes place predominantly via peritumoural lymphatic vessels. The weak expression of lymphangiogenic cytokines in neoplastic cells and lymphatic vessel proliferation in peritumoural regions and chronic pancreatitis indicate that inflammation may be the reason for the low rate of lymphangiogenesis.

Adult↗

PubMed2005

Role of CXCR3 in cellular but not humoral renal allograft rejection.

CXCR3, a chemokine receptor mainly expressed by T cells, is involved in animal transplant models and in human allograft rejection. CXCR3 expression was localized in formalin-fixed, paraffin-embedded renal allograft biopsies without signs of rejection (C4d-negative, Banff 0, n = 16), with C4d deposits as a sign of humoral rejection (C4d-positive, Banff 0, n = 8), with cellular rejection (C4d-negative, Banff I, n = 7) and with signs of both cellular and humoral rejection (C4d-positive, Banff 1, n = 5). Small, round infiltrating cells were CXCR3-positive. A high number of these cells was present in biopsies with cellular rejection (independent of C4d deposition). CXCR3-positive cells diffusely infiltrated the interstitium, including the tubular epithelium (tubulitis). CXCR3 scores and the area of CXCR3 staining were significantly higher in cellular rejection, when compared to biopsies without rejection, and with deposition of C4d alone. CXCR3-positive cells infiltrate renal allografts during cellular rejection, whereas C4d deposition is not associated with the recruitment of these cells.

Complement C4b↗

PubMed2005

R-roscovitine (CYC202) alleviates renal cell proliferation in nephritis without aggravating podocyte injury.

BACKGROUND: Cyclin-dependent kinase (CDK) inhibition is a new therapeutic approach to proliferative glomerulonephritides. CDK2 is required for G(1)/S transition and DNA synthesis and is inhibited by CYC202 (R-roscovitine). Since podocytes express CDK2 in nephritis and since loss of podocytes contributes to glomerulosclerosis, the rationale of the present study was to test whether CDK2 inhibition is safe in instances of podocyte injury. METHODS: Rats with passive Heymann nephritis, a model of membranous glomerulonephritis, were treated (day 3 to 30) with vehicle, low (25 mg/kg/day), or high (50 mg/kg/day) doses of CYC202. RESULTS: On day 27, blood pressure was normal in nephritic controls and was dose-dependently reduced by CYC202. Urinary albumin excretion did not differ between the groups on days 9, 16, 23, and 30. To investigate podocyte injury, we assessed the glomerular de novo expression of desmin, which was markedly up-regulated in almost all passive Heymann nephritis glomeruli but was not significantly different between the three groups. No tubulointerstitial de novo expression of desmin or alpha-smooth muscle actin (alpha-SMA), or tubulointerstitial monocyte/macrophage infiltration was noted in any group. Biologic activity of CYC202 was evident in the form of a dose-dependent decrease in the number of glomerular and tubulointerstitial mitotic figures as compared to vehicle alone. Glomerular immunostaining for cyclin D1, a marker for G(0) to G(1) transition, was significantly decreased in CYC202 treated groups at day 9. CONCLUSION: Whereas inhibition of CDKs by CYC202 reduced intrarenal cell proliferation in passive Heymann nephritis it did not aggravate podocyte damage, suggesting that this novel therapeutic approach is safe in renal diseases characterized by podocyte injury.

Animals↗

PubMed2005

The crucial role of macrophages in lymphangiogenesis.

Lymphangiogenesis is associated with pathological processes such as the metastatic spread of carcinoma cells and organization of immunologically active lymphocytic infiltrates following organ transplantation. It has not yet been established whether expansion of the lymphatic vascular meshwork is driven by incorporation of progenitor cells or by local endothelial cell division. In this issue of the JCI, Maruyama et al. provide evidence that after mouse corneal transplant, CD11b+ macrophages infiltrate the corneal stroma and transdifferentiate into lymphatic endothelial cell clusters that join existing lymphatic vessels. In complementary in vitro experiments, murine peritoneal macrophages expressed lymphatic endothelial markers and formed vessel-like protrusions. These findings add yet another facet to the plasticity of macrophages, which are already known to transform from naive monocytes into VEGF-C-producing cells. Thus, macrophages support lymphangiogenesis in 2 different ways, either by transdifferentiating and directly incorporating into the endothelial layer or by stimulating division of preexistent local lymphatic endothelial cells.

Animals↗

PubMed2005

The human glomerular podocyte is a novel target for insulin action.

Microalbuminuria is significant both as the earliest stage of diabetic nephropathy and as an independent cardiovascular risk factor in nondiabetic subjects, in whom it is associated with insulin resistance. The link between disorders of cellular insulin metabolism and albuminuria has been elusive. Here, we report using novel conditionally immortalized human podocytes in vitro and human glomeruli ex vivo that the podocyte, the principal cell responsible for prevention of urinary protein loss, is insulin responsive and able to approximately double its glucose uptake within 15 min of insulin stimulation. Conditionally immortalized human glomerular endothelial cells do not respond to insulin, suggesting that insulin has a specific effect on the podocyte in the glomerular filtration barrier. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1 and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. Insulin responsiveness in this key structural component of the glomerular filtration barrier may have central relevance for understanding of diabetic nephropathy and for the association of albuminuria with states of insulin resistance.

Actins↗