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.     

Evaluation of safety of endoscopic biopsy without cessation of antithrombotic agents in Japan

Background

Although guidelines in Japan recommend the cessation of antithrombotic agents before endoscopic biopsy, the safety of biopsy without the cessation of these agents has not been evaluated to date in this country. Therefore, we aimed to assess the feasibility of biopsy without cessation of antithrombotic agents in Japan.

Methods

This was a prospective single-arm study from a single institution. From May 2010 to November 2011, 112 outpatients who were receiving antithrombotic agents because of their high-risk status for a thromboembolic event (after implantation of coronary stent, after valve replacement, or a previous history of thromboembolic event or heart failure due to atrial fibrillation) were enrolled. We evaluated the rate of severe bleeding complications within 2?weeks after endoscopy and the endoscopic bleeding time (EBT) after biopsy in patients who underwent biopsy for endoscopic findings requiring pathology assessment.

Results

Among the 112 participants, 101 biopsies were performed for 48 and 12 outpatients who had had esophagogastroduodenoscopy and colonoscopy, respectively. All the biopsies provided enough specimens to evaluate pathologically. Hemostasis after biopsy was confirmed for all biopsies during endoscopic observation. No patients complained of any bleeding symptoms in the 2-week observation period after biopsy (0/101; 95% confidence interval [CI] 0–3.6%). Concerning the EBT (median 2.2?±?1.8?min, range 0.5–9?min), there were no significant differences between patients receiving single antithrombotic agents and those receiving multiple agents (2.4?±?1.4 vs. 2.1?±?2.1?min), nor were there any significant differences between patients not receiving and receiving warfarin (2.3?±?1.8 vs. 2.2?±?1.8?min).

Conclusion

Biopsy without cessation of antithrombotic agents, as recommended in Western guidelines, can also be acceptable for Japanese people if performed carefully.     

Diagnostic yield of dual-phase computed tomography enterography in patients with obscure gastrointestinal bleeding and a non-diagnostic capsule endoscopy

Background and Aim: In patients with obscure gastrointestinal (GI) bleeding, capsule endoscopy is widely used to determine the source of bleeding. However, there is currently no consensus on how to further evaluate patients with obscure GI bleeding with a non‐diagnostic capsule endoscopy examination. This study aims to determine the diagnostic yield of dual‐phase computed tomographic enterography (CTE) in patients with obscure GI bleeding and a non‐diagnostic capsule endoscopy. Methods: Patients with obscure GI bleeding who were referred for capsule endoscopy were prospectively enrolled. Obscure GI bleeding was defined as overt if there was obvious GI bleeding; otherwise it was defined as occult. Patients with a non‐diagnostic capsule endoscopy and no contraindications underwent a CTE. Results: Capsule endoscopy was performed in 52 patients; 26 patients (50%) had occult GI bleeding and 26 patients (50%) had overt GI bleeding. CTE was then performed in 25 of the 48 patients without a definitive source of bleeding seen on capsule endoscopy. The diagnostic yield of CTE was 0% (0/11) in patients with occult bleeding versus 50% (7/14) in patients with overt bleeding (P < 0.01). Using clinical follow up as the gold standard, for the 25 patients with a non‐diagnostic capsule, CTE had a sensitivity of 33% (95% confidence interval 0.15, 0.56) and a specificity of 75% (95% confidence interval 0.22, 0.99). Conclusions: In patients with a non‐diagnostic capsule endoscopy examination, CTE is useful for detecting a source of GI bleeding in patients with overt, but not occult, obscure GI bleeding.     

Genetic analysis of PRRT2 for benign infantile epilepsy,infantile convulsions with choreoathetosis syndrome,and benign convulsions with mild gastroenteritis

Purpose: PRRT2 mutations were recently identified in benign familial infantile epilepsy (BFIE) and infantile convulsions with paroxysmal choreoathetosis (ICCA) but no abnormalities have so far been identified in their phenotypically similar seizure disorder of benign convulsions with mild gastroenteritis (CwG), while mutations in KCNQ2 and KCNQ3 have been recognized in benign familial neonatal epilepsy (BFNE). The aim of this study was to identify PRRT2 mutations in infantile convulsions in Asian families with BFIE and ICCA, CwG and BFNE. Methods: We recruited 26 unrelated Japanese affected with either BFIE or non-familial benign infantile seizures and their families, including three families with ICCA. A total of 17 Japanese and Taiwanese with CwG, 50 Japanese with BFNE and 96 healthy volunteers were also recruited. Mutations of PRRT2 were sought using direct sequencing. Results: Heterozygous truncation mutation (c.649dupC) was identified in 15 of 26 individuals with benign infantile epilepsy (52.1%). All three families of ICCA harbored the same mutation (100%). Another novel mutation (c.1012+2dupT) was found in the proband of a family with BFIE. However, no PRRT2 mutation was found in either CwG or BFNE. Conclusions: The results confirm that c.649dupC, a truncating mutation of PRRT2, is a hotspot mutation resulting in BFIE or ICCA regardless of the ethnic background. In contrast, PRRT2 mutations do not seem to be associated with CwG or BFNE. Screening for PRRT2 mutation might be useful in early-stage differentiation of BFIE from CwG.     

Elevated urinary β2 microglobulin in the first identified Japanese family afflicted by X-linked myopathy with excessive autophagy

Here we report what is to our knowledge the first identified Japanese family afflicted by X-linked myopathy with excessive autophagy. The index case is a 52-year-old man with almost 40 years of progressive proximal muscle weakness. High urinary β2 microglobulin, normal serum β2 microglobulin, autophagic vacuoles with sarcolemmal features, and a hemizygous c.164–7T>G mutation in the VMA21 gene were found. His two maternal uncles had similar clinicopathological findings. High urinary β2 microglobulin without obvious renal dysfunction might result from decreased urine acidification in the distal convoluted tubules caused by the VMA21 gene mutation. These findings might prove to be useful as a preliminary marker suggestive of X-linked myopathy with excessive autophagy.