Structural Perspectives on Metal Dependent Roles of Ferric Uptake Regulator (Fur).

Iron is crucial for the metabolism and growth of most prokaryotic cells. The ferric uptake regulator (Fur) protein plays a central role in regulating iron homeostasis and metabolic processes in bacteria. It ensures the proper utilization of iron and the maintenance of cellular functions in response to environmental cues. Fur proteins are composed of an N-terminal DNA-binding domain (DBD) and a C-terminal dimerization domain (DD), typically existing as dimers in solution. Fur proteins have conserved metal-binding sites named S1, S2, and S3. Among them, site S2 serves as a regulatory site, and metal binding at S2 results in conformational changes. Additionally, as a transcriptional regulator, Fur specifically binds to a consensus DNA sequence called the Fur box. To elucidate the structural and functional properties of Fur proteins, various structures of metal- or DNA-bound Fur proteins or apo-Fur proteins have been determined. In this review, we focus on the structural properties of Fur proteins according to their ligand-bound state and the drug development strategies targeting Fur proteins. This information provides valuable insights for drug discovery.

Dual roles of myeloid-derived suppressor cells in various diseases: a review.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that originate from bone marrow stem cells. In pathological conditions, such as autoimmune disorders, allergies, infections, and cancer, normal myelopoiesis is altered to facilitate the formation of MDSCs. MDSCs were first shown to promote cancer initiation and progression by immunosuppression with the assistance of various chemokines and cytokines. Recently, various studies have demonstrated that MDSCs play two distinct roles depending on the physiological and pathological conditions. MDSCs have protective roles in autoimmune disorders (such as uveoretinitis, multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, type 1 diabetes, autoimmune hepatitis, inflammatory bowel disease, alopecia areata, and systemic lupus erythematosus), allergies, and organ transplantation. However, they play negative roles in infections and various cancers. Several immunosuppressive functions and mechanisms of MDSCs have been determined in different disease conditions. This review comprehensively discusses the associations between MDSCs and various pathological conditions and briefly describes therapeutic approaches.

Incidence and predictors of dorsal comminution in older adults with low-energy distal radius fracture.

PURPOSE: To identify the incidence of dorsal comminution using computed tomography (CT) images and identify predictors of this phenomenon in older adults with low-energy distal radius fractures (DRFs). METHODS: A total of 150 patients aged > 50 years with fall-induced dorsally angulated DRFs were enrolled in this study. Patients were divided into two groups based on the presence of dorsal comminution, defined as a metaphyseal void of greater than one-third of the maximum posterior to anterior depth of the bone on at least three cuts in the sagittal plane on post-reduction CT images. Data on participants' basic demographics, including age, sex, body mass index (BMI), and AO classification of DRFs, were collected. Bone mineral density (BMD) was assessed using T-scores of the femoral neck, and cortical thickness of the distal radius was determined from plain post-reduction radiographs. Radiological parameters and combined ulnar fractures were measured on plain pre-reduction radiographs. RESULTS: Among study participants, 91 (61%) had dorsal comminution, whereas 59 (39%) had no dorsal comminution on CT images. Both patient groups were compared based on presence of dorsal comminution, and showed no significant differences in age, sex, BMI, BMD, or cortical thickness on radiographs. However, all radiological parameters were better in the no dorsal comminution group than in the dorsal comminution group, and the proportion of patients with combined ulnar fractures was higher in the dorsal comminution group. In the multivariate analysis, the presence of combined ulnar fractures was the only significant predictor of dorsal comminution (p = 0.029, odds ratio = 2.267, 95% confidence interval: 1.085-4.736). CONCLUSION: The incidence of dorsal comminution is relatively high in patients with low-energy DRFs aged > 50 years. In particular, the presence of combined ulnar fractures is closely associated with dorsal comminution of DRFs. Thus, surgeons should exercise caution when evaluating this phenomenon.

Preparation of Hot-Melt-Extruded Solid Dispersion Based on Pre-Formulation Strategies and Its Enhanced Therapeutic Efficacy.

In this study, an amorphous solid dispersion containing the poorly water-soluble drug, bisacodyl, was prepared by hot-melt extrusion to enhance its therapeutic efficacy. First, the miscibility and interaction between the drug and polymer were investigated as pre-formulation strategies using various analytical approaches to obtain information for selecting a suitable polymer. Based on the calculation of the Hansen solubility parameter and the identification of the single glass transition temperature (Tg), the miscibility between bisacodyl and all the investigated polymers was confirmed. Additionally, the drug-polymer molecular interaction was identified based on the comprehensive results of dynamic vapor sorption (DVS), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, and a comparison of the predicted and experimental values of Tg. In particular, the hydroxypropyl methylcellulose (HPMC)-based solid dispersions, which exhibited large deviation between the calculated and experimental values of Tg and superior physical stability after DVS experiments, were selected as the most appropriate solubilized bisacodyl formulations due to the excellent inhibitory effects on precipitation based on the results of the non-sink dissolution test. Furthermore, it was shown that the enteric-coated tablets containing HPMC-bisacodyl at a 1:4 ratio (w/w) had significantly improved in vivo therapeutic laxative efficacy compared to preparations containing un-solubilized raw bisacodyl in constipation-induced rabbits. Therefore, it was concluded that the pre-formulation strategy, using several analyses and approaches, was successfully applied in this study to investigate the miscibility and interaction of drug-polymer systems, hence resulting in the manufacture of favorable solid dispersions with favorable in vitro and in vivo performances using hot-melt extrusion processes.

Effects of Abdominal Massage for Preventing Acute Postoperative Constipation in Hip Fractures: A Prospective Interventional Study.

Background: This prospective randomized controlled study aimed to determine the effects of abdominal massage on constipation management in elderly patients with hip fractures. Methods: From August 2017 to December 2018, patients aged above 65 years with hip fractures (n = 88) were randomly assigned to a massage group that received a bowel massage (n = 48) or a control group that did not receive a bowel massage (n = 40). Patients in the bowel massage group received a bowel massage from a trained caregiver after breakfast at approximately 9:00 AM for an hour. On admission, 5 days after surgery, and on the day of discharge, the patient's normal and actual defecation pattern, stool consistency, and any problems with defecation were assessed through a structured interview. The questionnaire comprising the Bristol Stool Scale, patient assessment of constipation, time to defecation, medication for defecations, failure to defecate, cause of admission, admission period, and date of surgery were recorded. Statistical analyses were performed 5 days after surgery and on the day of discharge. Results: The mean age of the study cohort was 81.4 years (range, 65-99 years). The number of constipation remedies was significantly lower in the massage group than in the control group on postoperative day (POD) 5 and at discharge (9 vs. 15, p = 0.049 and 6 vs. 11, p = 0.039, respectively). The number of defecation failures was significantly lower in the massage group than in the control group (10 vs. 17, p = 0.028) on POD 5. However, the number of defecation failures at discharge was not significantly different between the two groups (p = 0.131). The development of postoperative ileus (p = 0.271) and length of hospital stay (p = 0.576) were not different between the groups. Conclusions: The number of constipation remedies was significantly lower in the massage group than in the control group on POD 5 and discharge, and the number of defecation failures was significantly lower in the massage group than in the control group on POD 5. Therefore, abdominal massage may be considered as an independent nursing initiative for constipation management.

Changes in Shoulder Trauma during the COVID-19 Pandemic: A South Korean Survey.

Background: This study aimed to investigate the changes in the incidence of shoulder trauma and surgery 1 year after the outbreak of coronavirus disease 2019 (COVID-19) with social restriction compared with 1 year before the pandemic. Methods: Patients managed in our orthopedic trauma center between February 18, 2020, and February 17, 2021 (COVID-19 period) for shoulder trauma were compared with patients managed for the same duration a year ago (non-COVID-19 period; February 18, 2019, to February 17, 2020). The incidence of shoulder trauma, surgery, and mechanism of injury were compared between these periods. Results: The total number of shoulder trauma cases was lower in the COVID-19 period than in the non-COVID-19 period, although the difference was not significant (160 vs. 180 cases, p = 0.278). In addition, traumatic shoulder surgeries decreased during the COVID-19 period (57 vs. 69 cases, p = 0.285). The incidence of shoulder trauma according to four diagnostic classifications (contusion, sprain/subluxation, fracture, and dislocation) and fracture/dislocation types did not differ between the periods. During the COVID-19 period, accidental falls outdoors (45 vs. 67, p = 0.038) and sports-related injuries (15 vs. 29, p = 0.035) significantly decreased, but accidental falls at home (52 vs. 37, p = 0.112) increased compared with those during the non-COVID-19 period, although the difference was not significant. The monthly incidence of shoulder trauma decreased 2 months after the first outbreak (significant in March, p = 0.019), then steadily increased and significantly decreased during the second outbreak (August, p = 0.012). However, the third outbreak (December, p = 0.077) had little effect on the incidence of shoulder trauma. The number of monthly traumatic shoulder surgeries showed a similar pattern to the monthly incidence of shoulder trauma. Conclusions: During the COVID-19 pandemic, annual shoulder trauma cases and surgeries decreased compared to those in the non-COVID-19 period, even though the difference was insignificant. The incidence of shoulder trauma and surgery was significantly reduced in the early COVID-19 period; however, the effect of the pandemic on orthopedic trauma practice was minimal after approximately half a year. Decreases in falls outdoors and sports-related injuries, but an increase in falls at home, were observed during the COVID-19 pandemic.

Age, Tear Size, Extent of Retraction, and Fatty Infiltration Associated With a High Chance of a Similar Rotator Cuff Tear in the Contralateral Shoulder Regardless of Symptoms in Patients Undergoing Cuff Repair in the Index Shoulder.

PURPOSE: To investigate the prevalence of a contralateral rotator cuff tear (RCT) in patients with a symptomatic RCT requiring repair and to determine whether findings from magnetic resonance imaging (MRI) of the affected shoulder can predict the presence of a contralateral tear. METHODS: Patients with atraumatic RCTs who had undergone arthroscopic repair between March 2019 and February 2021 were reviewed in this study. MRI of both shoulder joints was performed to evaluate the bilaterality of RCT. Demographic factors and MRI findings of index shoulders were assessed using logistic regression analysis to reveal any correlations with the presence of RCT in the contralateral shoulder. RESULTS: A total of 428 patients were enrolled in this study. When the affected shoulders had a posterosuperior rotator cuff (PSRC) or subscapularis tear including either an isolated or combined tear, 63.6% and 67.8% had the same tears on the contralateral side, respectively. A contralateral-side tear was found in 74.6% (185/248) of symptomatic cases and 44.8% (65/145) of asymptomatic cases, which represents a significant difference (P < .001). Logistic regression analysis revealed that age ≥67.5 years, tear size ≥17 mm, Goutallier grade ≥1.5, and Patte grade ≥1.5 were found to be indicative of potential contralateral PSRC tears. By contrast, the presence of a subscapularis tear in the affected shoulder was the only significant risk factor in predicting a potential subscapularis tear in the contralateral shoulder. CONCLUSIONS: Among patients with a symptomatic RCT requiring arthroscopic repair, 63.6% with a PSRC tear and 67.8% with a subscapularis tear in the affected shoulder were found to have a similar tear in the contralateral shoulder regardless of symptoms. Age, tear size, extent of retraction, fatty infiltration of PSRC tears, and the presence of subscapularis tears were identified as factors predictive of tears on the contralateral side. LEVEL OF EVIDENCE: Level IV, case series.

The effect of cerclage wiring with intramedullary nail surgery in proximal femoral fracture: a systematic review and meta-analysis.

PURPOSE: The aim of this study was to evaluate the utility of cerclage wiring with intramedullary nail surgery in the treatment of proximal femoral fractures. METHODS: MEDLINE, Embase, and Cochrane Library were systematically searched for studies that evaluated the impact of cerclage wiring on proximal femoral fractures published up to September 20, 2021. Pooled analysis identified differences in the (1) fracture healing profile, (2) perioperative variables, (3) complications, and (4) clinical outcome score between cerclage wiring and non-cerclage wiring. RESULTS: Fourteen studies involving 1,718 patients with proximal femoral fractures who underwent cephalomedullary nailing surgeries were included. The pooled analysis revealed a longer fracture union time in the non-cerclage wiring group than in the cerclage wiring group (mean difference [MD] = - 1.03 months; 95% confidence interval [CI]: - 1.47 to - 0.59; P < 0.001), and there was no difference in the nonunion rate. The operation time was longer in the cerclage wiring group (MD = 14.32 min; 95% CI: 6.42-22.22; P < 0.001), but there were no differences in blood loss and the rate of poor quality of reduction. Superficial and deep infection rates showed no difference between the groups, and the readmission rate also showed no difference. The cerclage wiring group had a higher Harris hip score than the non-cerclage wiring group (MD = 2.13; 95% CI: 0.77-3.49; P = 0.002). CONCLUSIONS: Intramedullary nailing with cerclage wiring is considered a useful treatment method for proximal femoral fractures. It enables anatomic reduction and stable fixation, thereby reducing union time and facilitating rapid functional recovery.

Mesenchymal Stem Cells Potentiate the Vasculogenic Capacity of Endothelial Colony-Forming Cells under Hyperglycemic Conditions.

Many studies have demonstrated a reduced number and vasculogenic capacity of endothelial colony-forming cells (ECFCs) in diabetic patients. However, whether the vasculogenic capacity of ECFCs is recovered or not when combined with pericyte precursors, mesenchymal stem cells (MSCs), under hyperglycemic conditions has not been studied. Thus, we investigated the role of MSCs in ECFC-mediated vascular formation under high-glucose conditions. The ECFCs and MSCs were treated with normal glucose (5 mM; NG) or high glucose (30 mM; HG) for 7 days. The cell viability, proliferation, migration, and tube formation of ECFCs were reduced in HG compared to NG. Interestingly, the ECFC+MSC combination after HG treatment formed tubular structures similar to NG-treated ECFCs+MSCs. An in vivo study using a diabetic mouse model revealed that the number of perfused vessels formed by HG-treated ECFCs+MSCs in diabetic mice was comparable with that of NG-treated ECFCs+MSCs in normal mice. Electron microscopy revealed that the ECFCs+MSCs formed pericyte-covered perfused blood vessels, while the ECFCs alone did not form perfused vessels when injected into the mice. Taken together, MSCs potentiate the vasculogenic capacity of ECFCs under hyperglycemic conditions, suggesting that the combined delivery of ECFCs+MSCs can be a promising strategy to build a functional microvascular network to repair vascular defects in diabetic ischemic regions.

Analysis of the Efficacy of Universal Screening of Coronavirus Disease with Antigen-Detecting Rapid Diagnostic Tests at Point-or-Care Settings and Sharing the Experience of Admission Protocol-A Pilot Study.

AIMS: To introduce the admission protocol of a COVID-19 specialized hospital outlined by the government, including the assessment of reverse transcription polymerase chain reaction (RT-PCR), low dose chest computed tomography (CT) and antigen-detecting rapid diagnostic test (Ag-RDT) for patient screening. MATERIALS AND METHODS: This was a retrospective cohort study of 646 patients who were admitted between December 2020, and February 2021, during the third wave of COVID-19 in Korea. Ag-RDT and RT-PCR were routinely performed on all patients who required admission, and low-dose chest CT was performed on high-risk patients with associated symptoms. Any patients with high-risk COVID-19 infection according to the Ag-RDT test were quarantined alone in a negative pressured room, and those with low-risk COVID-19 infection remained in the preemptive quarantine room with or without negative pressure. The diagnostic values of the Ag-RDT test and associated cycle threshold (Ct) values of the RT-PCR test were subsequently evaluated. RESULTS: In terms of the diagnostic value, the Ag-RDT for COVID-19 had a sensitivity of 68.3%, specificity of 99.5%, positive predictive value (PPV) of 90.3%, and negative predictive value (NPV) of 97.9%. For the 355 symptomatic patients with low-dose chest CT, the diagnostic values of combined evaluations had a sensitivity of 90.2%, specificity of 99.0%, PPV of 86.1%, and NPV of 99.3%. The cut-off Ct value for positive Ag-RDT was ≤25.67 for the N gene (sensitivity: 89.3%, specificity: 100%), which was regarded as a high viable virus in cell culture. There were no patients or medical staff who had COVID-19 in the hospital. CONCLUSION: Appropriate patient care was possible by definitive triage of the area, according to the symptoms and using diagnostic tests. Screening protocols, including the Ag-RDT test and low-dose chest CT, could be helpful in emergency point-of-care settings.

Preparation and Characterization of Fenofibrate Microparticles with Surface-Active Additives: Application of a Supercritical Fluid-Assisted Spray-Drying Process.

In this study, supercritical fluid-assisted spray-drying (SA-SD) was applied to achieve the micronization of fenofibrate particles possessing surface-active additives, such as d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), sucrose mono palmitate (Sucroester 15), and polyoxyethylene 52 stearate (Myrj 52), to improve the pharmacokinetic and pharmacodynamic properties of fenofibrate. For comparison, the same formulation was prepared using a spray-drying (SD) process, and then both methods were compared. The SA-SD process resulted in a significantly smaller mean particle size (approximately 2 µm) compared to that of unprocessed fenofibrate (approximately 20 µm) and SD-processed particles (approximately 40 µm). There was no significant difference in the effect on the particle size reduction among the selected surface-active additives. The microcomposite particles prepared with surface-active additives using SA-SD exhibited remarkable enhancement in their dissolution rate due to the synergistic effect of comparably moderate wettability improvement and significant particle size reduction. In contrast, the SD samples with the surface-active additives exhibited a decrease in dissolution rate compared to that of the unprocessed fenofibrate due to the absence of particle size reduction, although wettability was greatly improved. The results of zeta potential and XPS analyses indicated that the surface-active additive coverage on the surface layer of the SD-processed particles with a better wettability was higher than that of the SA-SD-processed composite particles. Additionally, after rapid depletion of hydrophilic additives that were excessively distributed on the surfaces of SD-processed particles, the creation of a surface layer rich in poorly water-soluble fenofibrate resulted in a decrease in the dissolution rate. In contrast, the surface-active molecules were dispersed homogeneously throughout the particle matrix in the SA-SD-processed microparticles. Furthermore, improved pharmacokinetic and pharmacodynamic characteristics were observed for the SA-SD-processed fenofibrate microparticles compared to those for the SD-processed fenofibrate particles. Therefore, the SA-SD process incorporating surface-active additives can efficiently micronize poorly water-soluble drugs and optimize their physicochemical and biopharmaceutical characteristics.

Advanced Xenograft Model with Cotransplantation of Patient-Derived Organoids and Endothelial Colony-Forming Cells for Precision Medicine.

Preclinical evaluation models have been developed for precision medicine, with patient-derived xenograft models (PDXs) and patient-derived organoids (PDOs) attracting increasing attention. However, each of these models has application limitations. In this study, an advanced xenograft model was established and used for drug screening. PDO and endothelial colony-forming cells (ECFCs) were cotransplanted in NRGA mice (PDOXwE) to prepare the model, which could also be subcultured in Balb/c nude mice. Our DNA sequencing analysis and immunohistochemistry results indicated that PDOXwE maintained patient genetic information and tumor heterogeneity. Moreover, the model enhanced tumor growth more than the PDO-bearing xenograft model (PDOX). The PDO, PDOXwE, and clinical data were also compared in the liver metastasis of a colorectal cancer patient, demonstrating that the chemosensitivity of PDO and PDOXwE coincided with the clinical data. These results suggest that PDOXwE is an improvement of PDOX and is suitable as an evaluation model for precision medicine.

CCL8 mediates crosstalk between endothelial colony forming cells and triple-negative breast cancer cells through IL-8, aggravating invasion and tumorigenicity.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with a poor prognosis for which no effective therapeutic measures are currently available. The present study aimed to investigate whether interactions with endothelial colony-forming cells (ECFCs) promote aggressive progression of TNBC cells. Herein, using an indirect co-culture system, we showed that co-culture increased the invasive and migratory phenotypes of both MDA-MB-231 TNBC cells and ECFCs. Through a cytokine antibody array and RT-PCR analysis, we revealed that co-culture markedly induced secretion of the chemokine C-C motif ligand (CCL)8 from ECFCs and that of interleukin (IL)-8 from MDA-MB-231 cells. CCL8 was crucial for ECFC-induced IL-8 secretion and invasion of MDA-MB-231 cells as well as for MDA-MB-231-enhanced MMP-2 secretion and angiogenesis of ECFCs. We suggest c-Jun as a transcription factor for CCL8-induced IL-8 expression in MDA-MB-231 cells. IL-8 was important for co-culture-induced CCL8 and MMP-2 upregulation and invasion of ECFCs. Notably, our findings reveal a positive feedback loop between CCL8 and IL-8, which contributes to the aggressive phenotypes of both ECFC and TNBC cells. Using an MDA-MB-231 cell-based xenograft model, we show that tumor growth and metastasis are increased by co-injected ECFCs in vivo. Increased expression of IL-8 was observed in tissues with bone metastases in mice injected with conditioned media from co-cultured cells. High IL-8 levels are correlated with poor recurrence-free survival in TNBC patients. Together, these results suggest that CCL8 and IL-8 mediate the crosstalk between ECFCs and TNBC, leading to aggravation of tumorigenicity in TNBC.

Differential Angiogenic Responses of Human Endothelial Colony-Forming Cells to Different Molecular Subtypes of Breast Cancer Cells.

OBJECTIVE: Triple negative breast cancer (TNBC) is one subtype of breast cancer. It is characterized by lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Compared with non-TNBC, TNBC is more aggressive, of higher grade, and frequently metastatic with poor prognosis, which is correlated with upregulated microvascular density. Endothelial colony-forming cells (ECFCs) mediate neovascularization, which is the crucial contributor to cancer growth and metastasis. The present study aimed to determine whether angiogenic responses of ECFCs are regulated differently by TNBC compared with non-TNBC. METHODS: MDA-MB-231 and MCF7 cells were utilized for TNBC and non-TNBC, respectively. Bone-marrow-derived human ECFCs were treated with a conditioned medium (CM) of cancer cells to investigate the paracrine effect on angiogenesis. Also, ECFCs were co-cultured with cancer cells to evaluate the angiogenic effect of direct cell-to-cell interaction. Angiogenic responses of ECFCs were evaluated by proliferation, migration, and tube formation. Gene expression profiles of pro-angiogenic factors were also analyzed. RESULTS: Migration and tube formation of ECFCs were increased by treatment with CM of MDA-MB-231, which correlated with a higher gene expression profile of pro-angiogenic factors in MDA-MB-231 compared to MCF7. Interestingly, ECFCs co-cultured with MDA-MB-231 showed further increase of tube formation, suggesting synergic mechanisms between the paracrine effect and direct interaction between the cells. CONCLUSION: The angiogenic potential of ECFCs was enhanced by TNBC through both direct and indirect mechanisms. Therefore, the investigation of signaling pathways to regulate ECFC-mediated angiogenesis will be important to the discovery of anti-angiogenic therapies to treat TNBC patients.

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis.

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). in vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a 2×2 stitched area from the 4× object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, IC50 values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

Superoxide Dismutase Activity in Small Mesenteric Arteries Is Downregulated by Angiotensin II but Not by Hypertension.

Many studies reported reduced antioxidant capacity in the vasculature under hypertensive conditions. However, little is known about the effects of antihypertensive treatments on the regulation of vascular antioxidant enzymes. Thus, we hypothesized that antihypertensive treatments prevent the reduction of antioxidant enzyme activity and expression in the small vessels of angiotensin II-induced hypertensive rats (ANG). We observed the small mesenteric arteries and small renal vessels of normotensive rats (NORM), ANG, and ANG treated with a triple antihypertensive therapy of reserpine, hydrochlorothiazide, and hydralazine (ANG + TTx). Systolic blood pressure was increased in ANG, which was attenuated by 2 weeks of triple therapy (127, 191, and 143 mmHg for NORM, ANG, and ANG + TTx, respectively; p < 0.05). Total superoxide dismutase (SOD) activity in the small mesenteric arteries of ANG was lower than that of NORM. The protein expression of SOD1 was lower in ANG than in NORM, whereas SOD2 and SOD3 expression was not different between the groups. Reduced SOD activity and SOD1 expression in ANG was not restored in ANG + TTx. Both SOD activity and SOD isoform expression in the small renal vessels of ANG were not different from those of NORM. Interestingly, SOD activity in the small renal vessels was reduced by TTx. Between groups, there was no difference in catalase activity or expression in both the small mesenteric arteries and small renal vessels. In conclusion, SOD activity in the small mesenteric arteries decreased by angiotensin II administration, but not by hypertension, which is caused by decreased SOD1 expression.

Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells.

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an in vitro 3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the IC50 of vatalanib in ECFC+MSC spheroids at 24 h was 4.0 ± 0.40 µM, which are more correlated with the data of previous animal studies when compared with ECFC spheroids (0.2 ± 0.03 µM). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle.

Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.

Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects.

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.