Stem cell therapies benefit Alport syndrome.

Patients with Alport syndrome progressively lose renal function as a result of defective type IV collagen in their glomerular basement membrane. In mice lacking the alpha3 chain of type IV collagen (Col4A3 knockout mice), a model for Alport syndrome, transplantation of wild-type bone marrow repairs the renal disease. It is unknown whether cell-based therapies that do not require transplantation have similar potential. Here, infusion of wild-type bone marrow-derived cells into unconditioned, nonirradiated Col4A3 knockout mice during the late stage of disease significantly improved renal histology and function. Furthermore, transfusion of unfractionated wild-type blood into unconditioned, nonirradiated Col4A3 knockout mice improved the renal phenotype and significantly improved survival. Injection of mouse and human embryonic stem cells into Col4A3 knockout mice produced similar results. Regardless of treatment modality, the improvement in the architecture of the glomerular basement membrane is associated with de novo expression of the alpha3(IV) chain. These data provide further support for testing cell-based therapies for Alport syndrome.

The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.

A role of the protein Cbl in FGF-2-induced angiogenesis in murine brain endothelial cells.

The Cbl protein functions both as a multivalent adaptor and a negative regulator of receptor tyrosine kinases (RTKs), the latter by directing polyubiquitination of RTKs. To study the function of Cbl in endothelial cell signalling and angiogenesis, wild-type Cbl and tyrosine kinase binding (TKB) domain mutated Cbl (G306E) were overexpressed in murine immortalised brain endothelial (IBE) cells. Wild-type Cbl cells exhibited enhanced proliferation in low serum compared with the control and G306E Cbl cells. Furthermore, up-regulated phosphorylation of fibroblast growth factor receptor 1 (FGFR-1) and Akt were observed in wild-type Cbl cells upon FGF-2 stimulation. A Cbl TKB domain mutant, G306E, disrupted the phosphorylation of the FGFR-1 but not that of FRS2. In the tubular morphogenesis assay, cells expressing wild-type Cbl initially formed tubular structures. These showed decreased stability and converted into cell aggregates, possibly due to a failure to cease proliferating. Our data support the idea that the wild-type Cbl cells exhibit enhanced proliferation, and thus lose their ability to differentiate appropriately. The present study reveals a role of the Cbl protein in FGF-2 dependent signalling in endothelial cells by its destabilisation of tubular structures.

The role of the adapter protein SHB in embryonic stem cell differentiation into the pancreatic beta-cell and endothelial lineages.

Embryonic stem (ES) cells represent an attractive tool not only for the study of the development of various cell types but also as a potential source of cells for transplantation. Previous studies suggested a role of the signal transduction protein SRC homology 2(SH2) protein of Beta-cells (SHB) for the development of both pancreatic 3-cells and blood vessels. SHB is an SH2 domain-containing adapter protein involved in the generation of signaling complexes in response to activation of a variety of receptors, several of which have been implicated in developmental processes. Moreover, microarray analysis of ES cells expressing mutant SHB has revealed decreased expression of several genes of developmental importance. Here, we present protocols that may be used for transfection of mouse ES cells and to study the differentiation of ES cell-derived embryoid bodies (EBs) into the pancreatic Beta-cell lineage as well as into vascular structures with special reference to the effect of SHB. Moreover, we also provide a protocol that may be used for enrichment by fluorescence-activated cell sorting of specific cell lineages in EBs.

Increased endothelial progenitor cells and vasculogenic factors in higher-staged arteriovenous malformations.

UNLABELLED: ACKGROUND:: Arteriovenous malformations cause significant morbidity, primarily because they expand over time and recur after treatment. The authors hypothesized that neovascularization might contribute to arteriovenous malformation progression. METHODS: Arteriovenous malformation tissue was collected prospectively from 12 patients after resection. Schobinger stage was determined by clinical history. Neovascularization in stage II lesions (n=7) was compared with stage III arteriovenous malformations (n=5) that had progressed. Specimens were analyzed using immunohistochemistry for CD31, Ki67, and CD34/CD133. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to determine mRNA expression of factors that recruit endothelial progenitor cells: vascular endothelial growth factor (VEGF), stromal cell-derived factor-1α (SDF-1α), and hypoxia-inducible factor-1α (HIF-1α). VEGF receptors (VEGFR1, VEGFR2, neuropilin 1, and neuropilin 2) also were quantified using quantitative real-time reverse-transcriptase polymerase chain reaction. RESULTS: Stage III arteriovenous malformations showed greater microvessel density (5.8 percent) than stage II lesions (1.3 percent) (p=0.004); no difference in proliferating endothelial cells was noted (p=0.67). CD133CD34 endothelial progenitor cells were elevated in stage III (0.53 percent) compared with stage II arteriovenous malformations (0.25 percent) (p=0.03). HIF-1α and SDF-1α were increased 7.6- and 7.9-fold in stage III compared with stage II lesions (1.7-fold and 3.3-fold), respectively (p=0.02). Neuropilin 1 and neuropilin 2 expression was greater in stage III (5.8-fold and 4.6-fold) than stage II arteriovenous malformations (3.0-fold and 2.4-fold) (p=0.03). CONCLUSIONS: Higher-staged arteriovenous malformations exhibit increased expression of endothelial progenitor cells and factors that stimulate their recruitment. Neovascularization by vasculogenesis may be involved in progression of arteriovenous malformation.

Induction of lymphatic endothelial cell differentiation in embryoid bodies.

The molecular mechanisms that regulate the formation of the lymphatic vascular system remain poorly characterized. Whereas studies in embryonic stem (ES) cells have provided major new insights into the mechanisms of blood vessel formation, the development of lymphatic endothelium has not been previously observed. We established embryoid bodies (EBs) from murine ES cells in the presence or absence of lymphangiogenic growth factors. We found that lymphatic endothelial cells develop at day 18 after EB formation. These cells express CD31 and the lymphatic lineage markers Prox-1 and Lyve-1, but not the vascular marker MECA-32, and they frequently sprout from preexisting blood vessels. Lymphatic vessel formation was potently promoted by VEGF-A and VEGF-C but not by bFGF. Our results reveal, for the first time, that ES cells can differentiate into lymphatic endothelial cells, and they identify the EB assay as a powerful new tool to dissect the molecular mechanisms that control lymphatic vessel formation.

Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta signaling.

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.

Role of the Src homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation.

To study the role of the Src homology 2 (SH2) domain-containing protein Shb in angiogenesis, wild-type Shb and SH2 domain-mutated Shb (R522K Shb) were overexpressed in murine immortalized brain endothelial cells. The wild-type Shb cells exhibited an increased rate of apoptosis on serum withdrawal. Both wild-type Shb and R522K Shb cells exhibited enhanced spreading concomitant with cytoskeletal rearrangements that occurred independently of fibroblast growth factor (FGF)-2 stimulation. However, these effects may partly be caused by altered regulation of Rac1 and Rap1 activation in the Shb cells. The Shb-induced cytoskeletal rearrangements were not dependent on phosphatidylinositol 3' kinase activity, but could be reversed by inhibition of Src family kinases. FGF-2 failed to further enhance migration of wild-type Shb and R522K Shb cells. The R522K Shb cells cultured in collagen gels exhibit diminished tubular morphogenesis when treated with FGF-2, implicating the need for a functional Shb molecule in this process. These data suggest that Shb plays a role in the proliferation and differentiation of endothelial cells and, hence, participates in angiogenesis.