Direct Isolation, Culture and Transplant of Mouse Skeletal Muscle Derived Endothelial Cells with Angiogenic Potential

Background: Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study. Methodology: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1 +, CD31 +, CD34 dim and CD45 2 cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature. Conclusion: This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications

Dystrophin‐deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154288/1/fsb2fj067353com.pd

Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues

Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis

Tick saliva is a rich source of modulators of vascular biology. We have characterized Ixonnexin, a member of the “Basic-tail” family of salivary proteins from the tick Ixodes scapularis. Ixonnexin is a 104 residues (11.8 KDa), non-enzymatic basic protein which contains 3 disulfide bonds and a C-terminal rich in lysine. It is homologous to SALP14, a tick salivary FXa anticoagulant. Ixonnexin was produced by ligation of synthesized fragments (51–104) and (1–50) followed by folding. Ixonnexin, like SALP14, interacts with FXa. Notably, Ixonnexin also modulates fibrinolysis in vitro by a unique salivary mechanism. Accordingly, it accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) with Km 100 nM; however, it does not affect urokinase-mediated fibrinolysis. Additionally, lysine analogue ε-aminocaproic acid inhibits Ixonnexin-mediated plasmin generation implying that lysine-binding sites of Kringle domain(s) of plasminogen or t-PA are involved in this process. Moreover, surface plasmon resonance experiments shows that Ixonnexin binds t-PA, and plasminogen (KD 10 nM), but not urokinase. These results imply that Ixonnexin promotes fibrinolysis by supporting the interaction of plasminogen with t-PA through formation of an enzymatically productive ternary complex. Finally, in vivo experiments demonstrates that Ixonnexin inhibits FeCl3-induced thrombosis in mice. Ixonnexin emerges as novel modulator of fibrinolysis which may also affect parasite-vector-host interactions

Neurotrophin-3 is a novel angiogenic factor capable of therapeutic neovascularization in a mouse model of limb ischemia

OBJECTIVE: To investigate the novel hypothesis that neurotrophin-3 (NT-3), an established neurotrophic factor that participates in embryonic heart development, promotes blood vessel growth. METHODS AND RESULTS: We evaluated the proangiogenic capacity of recombinant NT-3 in vitro and of NT-3 gene transfer in vivo (rat mesenteric angiogenesis assay and mouse normoperfused adductor muscle). Then, we studied whether either transgenic or endogenous NT-3 mediates postischemic neovascularization in a mouse model of limb ischemia. In vitro, NT-3 stimulated endothelial cell survival, proliferation, migration, and network formation on the basement membrane matrix Matrigel. In the mesenteric assay, NT-3 increased the number and size of functional vessels, including vessels covered with mural cells. Consistently, NT-3 overexpression increased muscular capillary and arteriolar densities in either the absence or the presence of ischemia and improved postischemic blood flow recovery in mouse hind limbs. NT-3–induced microvascular responses were accompanied by tropomyosin receptor kinase C (an NT-3 high-affinity receptor) phosphorylation and involved the phosphatidylinositol 3-kinase–Akt kinase–endothelial nitric oxide synthase pathway. Finally, endogenous NT-3 was shown to be essential in native postischemic neovascularization, as demonstrated by using a soluble tropomyosin receptor kinase C receptor domain that neutralizes NT-3. CONCLUSION: Our results provide the first insight into the proangiogenic capacity of NT-3 and propose NT-3 as a novel potential agent for the treatment of ischemic disease

Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury

Background: Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described. Methodology/Principal Findings: In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with a7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury. Conclusions/Significance: Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration

HTLV-1 infection in acute t- lymphocytic leukemia/lymphoma

Adult T- lymphocyte leukemia/ lymphoma (ATLL), described by Uchiyama et al. in 1977, is a distinct neoplasia of peripheral T-lymphocytes caused by human T-cell lymphotropic virus type 1 (HTLV-1). The authors describe the case of a 75-year-old female patient who presented with fever, chills, and altered mental status. The peripheral blood morphology showed large atypical lymphocytes with multilobed nuclei and flow cytometry consistent with ATLL. The authors discuss the pathophysiology, differential diagnosis, and subtypes of ATLL in addition to the diagnostic approach using flow cytometry when bone marrow biopsy is not available and modalities of treatment

EC injected subcutaneously in matrigel migrate into the muscle.

<p>Top, a montage of the matrigel plug shows PKH26⁺ microvessels in the matrigel plug over the TA muscle. Bottom, A montage of an area approximately 80 µm distal shows migration of PKH26⁺ EC. EC invaded the TA from the matrigel plug and formed new microvessels along their migration path and also integrated into existing vessel. Staining with anti-vWF in Alexa 647 (magenta) highlights EC angiogenic capability within the matrigel plug and muscle. Scale bars = 50 µm.</p