Progressive Bundling of Fibrillin Microfibrils into Oxytalan Fibers in the Chick Presumptive Dermis

Dorsoventral fibers in the presumptive dermis of the chick limb bud reported first by Hurle's group in 1989 are now revealed as bundles of fibrillin microfibrils (Isokawa et al., 2004). The bundles, which could be called oxytalan fibers at the light microscopic level, are aligned perpendicularly to the overlying ectoderm and form a unique fiber array, originating directly from the basal lamina. This well‐oriented organization is beneficial in examining the process of in vivo bundling of microfibrils into oxytalan fibers. In this study, sections through the presumptive limb dermis were preferentially prepared from chick embryos at Days 4–6 (ED4‐6). Immunohistochemically, fibrillin‐positive dots representing cross‐sectioned surfaces of individual fibers, increased in size from ED4 to 6, but their number per unit area remained constant. Ultrastructurally, a single oxytalan fiber at ED4 consisted of ～15 microfibrils; the latter number increased fourfold from ED4 to 5 and threefold from ED5 to 6. Oxytalan fibers were all closely associated with mesenchymal cell; notably, the fibers at ED5 and 6 were held in a shallow ditch on the cell body or by lamellipodial cytoplasmic protrusion. In the sites of cell–fiber adhesion, microfibrils in the periphery of an oxytalan fiber appeared to adhere directly or by means of short flocculent strands to a nearby cell membrane; the latter showed a thickening of plasmalemma and its undercoat, indicating the presence of adhesive membrane specification. These findings suggest that the bundling of microfibrils is a progressive and closely cell‐associated process. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

FER overexpression is associated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer

Here, we show that overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, predicts poor postoperative outcome and might be involved in cancer-cell survival in non-small cell lung cancer (NSCLC). Systematic screening using in silico analyses and quantitative RT-PCR revealed that FER was overexpressed in about 10% of NSCLC patients. Evaluation of FER expression using immunohistochemistry (IHC) on tissue microarrays was consistent with the mRNA level detected using quantitative RT-PCR. In analyses of 135 NSCLC patients who had undergone potential curative resection, we found that FER overexpression detected using IHC had no association with clinicopathological features such as age, sex, smoking history, histological type, disease stage, T factor, N factor, adjuvant chemotherapy history, or EGFR mutation, but was correlated with poor postoperative survival periods. A multivariate Cox regression analysis showed that this prognostic impact was independent of other clinicopathological features. In functional analyses of FER in vitro, FER exhibited a transforming activity, suggesting that it possesses oncogenic functions. We also found that human lung cancer NCI-H661 cells, which exhibited FER-outlier expression, were led to apoptosis by the knockdown of FER using RNA interference. FER overexpression might serve as a prognostic biomarker and be involved in cancer-cell survival in NSCLC.     

Quality-Control Culture System Restores Diabetic Endothelial Progenitor Cell Vasculogenesis and Accelerates Wound Closure

Delayed diabetic wound healing is, in part, the result of inadequate endothelial progenitor cell (EPC) proliferation, mobilization, and trafficking. Recently, we developed a serum-free functional culture system called the quality and quantity culture (QQc) system that enhances the number and vasculogenic potential of EPCs. We hypothesize that QQc restoration of diabetic EPC function will improve wound closure. To test this hypothesis, we measured diabetic c-kit⁺Sca-1⁺lin⁻ (KSL) cell activity in vitro as well as the effect of KSL cell–adoptive transfer on the rate of euglycemic wound closure before and after QQc. KSL cells were magnetically sorted from control and streptozotocin-induced type I diabetic C57BL6J bone marrow. Freshly isolated control and diabetic KSL cells were cultured in QQc for 7 days and pre-QQc and post-QQc KSL function testing. The number of KSL cells significantly increased after QQc for both diabetic subjects and controls, and diabetic KSL increased vasculogenic potential above the fresh control KSL level. Similarly, fresh diabetic cells form fewer tubules, but QQc increases diabetic tubule formation to levels greater than that of fresh control cells (P < 0.05). Adoptive transfer of post-QQc diabetic KSL cells significantly enhances wound closure compared with fresh diabetic KSL cells and equaled wound closure of post-QQc control KSL cells. Post-QQc diabetic KSL enhancement of wound closure is mediated, in part, via a vasculogenic mechanism. This study demonstrates that QQc can reverse diabetic EPC dysfunction and achieve control levels of EPC function. Finally, post-QQc diabetic EPC therapy effectively improved euglycemic wound closure and may improve diabetic wound healing.Although blood supply is essential for tissue viability, new blood vessel formation is critical for tissue recovery, regeneration, and repair. Postnatal new blood vessel formation was long thought to be restricted to angiogenesis, the sprouting of new blood vessels from existing vascular structures. However, in 1997, we demonstrated that the de novo formation of new blood vessel derived from bone marrow (BM)-derived cells (i.e., vasculogenesis) is an important part of postnatal healing (1–3). The BM-derived endothelial progenitor cells (EPCs) are precursors of endothelial cells (ECs) and are characterized by their surface expression of KDR, CD133, and CD34 for humans and of lineage-negative c-kit⁺Sca-1⁺ (KSL) cells for murine BM cells (4–6).After injury, locally derived circulating factors mobilize EPCs from their endosteal BM niche. Circulating BM-derived EPCs traffic to the site of injury, experience diapedesis, cluster, tubulize, and canalize to form nascent vessels that inosculate with the existing vasculature (7,8). EPCs have been shown to revascularize numerous ischemic tissues, including myocardium (i.e., myocardial infarction), brain (i.e., cerebral infarction), and skin (i.e., cutaneous wounding) (9,10). Whereas BM-derived EPCs contribute to only 25% of newly formed endothelium in healing tissues, when EPC function is impaired there are marked deficits in tissue repair mechanisms (11,12).Compared with nondiabetic patients, diabetic EPCs have impaired proliferation, adhesion, migration, and differentiation (13–15). Although the pathogenesis of impaired diabetic wound healing is multifactorial, EPC dysfunction plays a central role (16,17). These intrinsic diabetic EPC vasculogenic impairments may result in >83,000 amputations each year and a postamputation 3-year mortality rate of 75.9% (18). In preclinical studies, the administration of exogenous EPCs has improved ventricular function after myocardial ischemia (19,20), enhanced neuronal recovery after cerebral vascular occlusion, and accelerated restoration of blood flow to ischemic limbs (13,16,17,21–23). Based on these exciting results, we have conducted a phase 3 clinical trial of autologous granulocyte colony-stimulating factor–mobilized peripheral blood EPC therapy for nonhealing diabetic foot patients (24). The results demonstrated that more successful therapeutic results were seen in patients receiving high-vasculogenic EPCs. From these results, we hypothesize that successful autologous diabetic EPC therapy relies on the vasculogenic function of transplanted EPCs and speculate that the intrinsic diabetic EPC dysfunction will limit the efficacy of the therapeutic strategy (25,26).Recently, our group established a serum-free quality and quantity culture (QQc) system (containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand) that enhances the vasculogenic potential of EPCs (27). We hypothesize that QQc can reverse the detrimental effects of diabeties-induced EPC dysfunction and can supply a sufficient number of functional EPCs for adoptive autologous cell–based therapy for diabetic patients. In the current study, we tested this hypothesis.