Intact and healthy hair follicles are important for hair growth after hair follicle transplantation. However, effective and practical evaluation methods for the quality of hair follicles are currently lacking. In the present study, we developed a novel fast staining method for histological examination of hair follicles. The whisker follicles from mice were used to explore the staining protocols, and the final protocol for the evaluation of human hair follicles was derived from animal experiments. After extraction, human hair follicles or mouse whisker follicles were permeabilized with 0.3% Triton X-100. Subsequently, hair follicles were processed by either hematoxylin or alkaline phosphatase staining. The integrity and growth state, including the status of hair follicle stem cells and blood vessels of the extracted hair follicles, were clearly identified under a light microscope. Unhealthy hair follicles from donors or hair follicles broken during extraction were easily revealed by this method. Importantly, it took less than half an hour to obtain images of an individual hair follicle. This method is simple and practical for evaluating the quality and status of hair follicles, providing a fast-screening procedure for hair follicle transplantation.

Myocardial ischemia leads to cardiac fibrosis along with copper (Cu) loss. Cu repletion diminishes myocardial fibrosis and improves cardiac function. The transformation of fibroblasts to myofibroblasts is highly responsible for the pathogenesis of cardiac fibrosis. This study was undertaken to test the hypothesis that Cu inhibition of cardiac fibrosis results from suppression of myofibroblasts. Rhesus monkeys 4-5 years old were subjected to coronary artery ligation to induce myocardial infarction (MI). At the end of the fourth week after the surgery, an ultrasound-directed Cu-albumin microbubble organ-specific Cu delivery technique was used to treat the ischemia-infarcted monkey hearts twice a week for 4 weeks. This treatment increased Cu concentrations in the infarct area, loosened the collagen cross-linking network, restored blood vessel density, and improved cardiac contractility. Total fibroblasts labeled with vimentin were increased in the infarct area, and Cu repletion did not alter this increase. Myofibroblasts, dually labeled with vimentin and α-smooth muscle actin (α-SMA), were also significantly increased in the infarct area but were significantly reduced by Cu repletion. Correspondingly, the products of myofibroblasts, type I and III collagens and inhibitors of collagenases were significantly reduced. In contrast, metalloproteinase-1 (MMP-1) and MMP-1 producing fibroblasts (vimentin+ and MMP-1+ cells) were significantly increased. These results suggest that Cu inhibits the transformation of fibroblasts to myofibroblasts, leading to a pro-fibrinolytic switch and an improvement in cardiac function.

Copper depletion is associated with myocardial ischemic infarction, in which copper metabolism MURR domain 1 (COMMD1) is increased. The present study was undertaken to test the hypothesis that the elevated COMMD1 is responsible for copper loss from the ischemic myocardium, thus worsening myocardial ischemic injury. Mice (C57BL/6J) were subjected to left anterior descending coronary artery permanent ligation to induce myocardial ischemic infarction. In the ischemic myocardium, copper reduction was associated with a significant increase in the protein level of COMMD1. A tamoxifen-inducible, cardiomyocyte -specific Commd1 knockout mouse (C57BL/6J) model ( COMMD1CMC▲/▲) was generated using the Cre-LoxP recombination system. COMMD1CMC▲/▲ and wild-type littermates were subjected to the same permanent ligation of left anterior descending coronary artery. At the 7th day after ischemic insult, COMMD1 deficiency suppressed copper loss in the heart, along with preservation of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 expression and the integrity of the vascular system in the ischemic myocardium. Corresponding to this change, infarct size of ischemic heart was reduced and myocardial contractile function was well preserved in COMMD1CMC▲/▲ mice. These results thus demonstrate that upregulation of COMMD1 is at least partially responsible for copper efflux from the ischemic heart. Cardiomyocyte-specific deletion of COMMD1 helps preserve the availability of copper for angiogenesis, thus suppressing myocardial ischemic dysfunction.

The present study was undertaken to investigate whether Cu protects vasculatures from ischemic injury in the heart. C57/B6 mice were introduced to myocardial ischemia (MI) by permanent ligation of the left anterior descending (LAD) coronary artery. Two hours post-LAD ligation, mice were intravenously injected with a Cu-albumin (Cu-alb) solution, or saline as control. At 1, 4, or 7 days post-MI, hearts were collected for further analysis. A dramatic decrease in CD31-positive endothelial cells concomitantly with abundant apoptosis, along with obstruction of blood flow, was observed in ischemic myocardium 1 day post-MI. The early Cu-alb treatment protected CD31-positive cells from apoptosis, along with a preservation of micro-vessels and a decrease in infarct size. This early vasculature preservation ensured myocardial blood perfusion and protected cardiac contractile function until 28 days post-MI. This strategy of Cu-alb treatment immediately following MI would help develop a therapeutic approach for acute heart attack patients in a clinical setting.

Previous studies have shown that cardiomyocytes in the sub‐endocardial region of infarct myocardium survive from ischemic injury. The present study was undertaken to explore a possible mechanism by which these cardiomyocytes survive in response to myocardial ischemia. Wild type mice (C57BL/6J, male, 8–10 weeks), or endothelial cell‐specific red fluorescent protein (RFP) expression transgenic mice (Tie2CRETdTomato) were subjected to permanent ligation of left anterior descending of coronary artery along with sham‐operated controls in which the same surgery procedure was applied with an exception of the artery ligation. Pimonidazole was injected intravenously to define the hypoxic region, and lectin was injected to trace blood infiltration before sacrifice in all of the mice. The hearts were harvested on day one after the surgery and cut into serial sections for detection of cardiomyocytes and endothelial cells. The staining of pimonidazole showed that most of the infarct myocardium was under hypoxic condition except the sub‐endocardial region in which survived cardiomyocytes were detected. The RFP positive endothelial cells in the sub‐endocardial region also survived but displayed a suppressed expression of PECAM‐1 and VEGFR2. Correspondently, lectin infiltration analysis suggested blood leakage in the sub‐endocardial region although there was no blood infiltration in other regions of the infarct area. The results suggest that blood infiltration through the transformed endothelial cells in the sub‐endocardial region of infarcted myocardium would provide a non‐hypoxic microenvironment, contributing to the survival of cardiomyocytes in this region.Support or Funding InformationWest China Hospital, Sichuan UniversityThe changes of hypoxic microenvironment in the infarct myocardium region.Hypoxic condition (partial pressure of O2 was less than 10 mmHg) was marked with hpi (pimonidazole) at 1st day after ischemic injury. The blood infiltrations were reflected by FITC‐conjugated WGA through tail vein at 1st, 4th, 7th day after LAD ligation. Hypoxic region stained by pimonidazole (red), and nuclear labeled by DAPI (blue). Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; MI, myocardial infarction.Figure 1The change of blood supplement in the sub‐endocardial region.The blood infiltrations were completely reflected by Lectin (FITC‐conjugated WGA) through tail vein at 1st day after LAD ligation. Cardiomyocytes stained by TNNI3 (red), Blood infiltration labeled by FITC‐conjugated WGA (green), and nuclear labeled by DAPI (blue). Abbreviations: WGA, wheat germ agglutinin; DAPI, 4′, 6‐diamidino‐2‐phenylindole; MI, myocardial infarction.Figure 2

Copper (Cu) supplementation improves myocardial structural and functional recoveries from chronic ischemic injury in both monkey and mouse models, and myocardial vascular regeneration plays a critical role in this process. The present study was undertaken to test the hypothesis that the homing and differentiation of stem cells make an important contribution to myocardial vascular regeneration in mouse model of ischemic heart disease. Male C57 mice of 8–12 weeks old were subjected to left anterior descending (LAD) coronary artery ligation to induce myocardial ischemic infarction. Four weeks after the surgery, a cardiac‐specific ultrasound‐mediated Cu‐albumin microbubble (Cu‐MB) Cu delivery procedure was used to supplement Cu to the heart twice a week for two weeks, and the controls were treated with the same procedure but with Cu‐absent albumin microbubble. All of the mice were then injected with PKH26‐labeled bone marrow‐derived stem cells (BMSCs), and the hearts were harvested at 1st, 3rd, 7th, or 14th day after the cell transplantation. One day after cell transplantation, the homing of PKH26‐labeled BMSCs in the ischemic infarct area was significantly increased in the Cu‐MB and ultrasound treated mice, but not in the ultrasound untreated mice. Three days after cell transplantation, the number of BMSC‐derived endothelial cells, as defined by the cells dually‐labeled with PKH26 and CD31 were significantly increased in the ischemic infarct area of the mice treated with ultrasound and Cu‐MB. The capillary densities in the ischemic infarct area of the mice treated with ultrasound and Cu‐MB were significantly elevated in comparison with that of mice treated with ultrasound and MB without Cu at 7th or 14th day after cell transplantation. Myocardial structure and contractile function were also significantly improved 14 days after cell transplantation in the ultrasound and Cu‐MB treated mice. These results thus demonstrate that Cu supplementation promotes myocardial vascular regeneration through at least in part enhancing stem cells homing and differentiation to vascular endothelial cells in the infarct myocardium, improving myocardial structural and functional recovery from chronic ischemic injury.Support or Funding InformationScientific research funds of West China Hospital, Sichuan UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Previous studies demonstrated that myocardial ischemia causes copper (Cu) reduction and Cu metabolism MURR domain 1 (COMMD1) protein accumulation in the ischemic infarct myocardium. Since COMMD1 is involved in the export of Cu from several types of cells, the present study was undertaken to test the hypothesis that the accumulation of COMMD1 is responsible for Cu reduction in the ischemic heart, thus worsening myocardial ischemic injury. We generated a tamoxifen‐induced, cardiac‐specific COMMD1 knockout mouse model ( Myh6‐cre ER COMMD1 flox/flox ) to examine the alterations in Cu status and myocardial function in mice subjected to ischemic insult. Six‐week‐old, male Myh6‐cre ER COMMD1 flox/flox and littermate control mice were injected intraperitoneally with tamoxifen for two weeks and subsequently subjected to left anterior descending (LAD) coronary artery ligation or sham operation. Cardiac function was determined by echocardiography on the seventh day after the operation. Histological examinations including hematoxylin and eosin stain (HE) and sirius red (SR) staining were performed to visualize the differences in infracted area, myocardial cell death, and fibrosis. The results showed that in the absence of COMMD1, ischemia‐induced reduction of Cu concentrations in the heart was alleviated, and the declines in both ejection fraction (EF) and fractional shortening (FS) were significantly suppressed. These changes were accompanied by the reduction in the infarct size, cell death, and fibrosis in the COMMD1‐deficient heart. These results thus suggest that COMMD1 plays a critical role in the pathogenesis of ischemic heart disease, and deletion of COMMD1 leads to suppression of myocardial ischemic injury. Support or Funding Information Scientific research funds of West China Hospital of Sichuan University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Rabbits are widely used for the study of atherosclerosis; however, the lack of a unified and quantitative analysis of atheroma limits data interpretation and comparisons between laboratories. In this study, we applied a simple quantitative method, referred to as the oil red O (ORO) dye-eluting method, for analysis of atherosclerotic plaques in freshly isolated aortas. It employs ORO staining of the plaques followed by elution of the dye that is subjected to quantitative measurement. Atherosclerosis was induced in rabbits by feeding a 1% (w/w) high cholesterol diet for 4 or 12 weeks. Thoracic aortas were isolated and sufficiently stained by ORO. These dyes were easily and completely extracted by 100% ethanol and quantified by spectrophotometric measurement at 510 nm. A series of cross-sectional slices at 100-µm intervals were counterstained by elastic van Gieson. It was found that there was a highly positive correlation between the dye concentration and the amount of plaque tissue, determined as volume of plaques (regression coefficient r2: 0.8792, p < 0.001). The color equivalence of the dye content was expressed as µg/mm2 of intimal aorta area to allow direct comparisons among aortas. The color equivalences of ORO content in rabbits fed 12 weeks were almost 5.0 times higher than those fed 4 weeks. Thus, this ORO dye-eluting method is useful for quantification of atherosclerotic plaques in aortas in rabbits, as well as other animal models.