Establishment of the improved colonization of Escherichia coli laboratory strain in the intestine mediated by single gene deletion.

In light of the emerging importance of the gut microbiome in human health, there is a need to improve the colonization efficiency of therapeutic bacteria called probiotics. Despite their recognized potential, artificially administered bacteria exhibit poor colonization in the intestine, limiting their therapeutic efficacy. Addressing this challenge requires innovative strategies; however, reported examples are limited. In nature, including in the intestinal tract, bacteria live via biofilm formation. Recently, it has been reported that RNase I, a member of the RNase T2 family conserved among almost all species, including bacteria, inhibits biofilm formation in Escherichia coli. In this study, we focus on these results and investigate the relationship between high biofilm formation and intestinal attachment using a non-settling E. coli laboratory strain as a probiotic model. The intestinal colonization abilities were evaluated through a microfluidic device mimicking the intestinal tract and through oral administration to mice. The in vitro and in vivo experiments showed that the E. coli strain lacking RNase I exhibited remarkable stability in intestinal colonization. We investigated the observation of colonization using fluorescence in situ hybridization, and inoculated E. coli cells were aggregated with the gut microbiome in the cecum and colon. This study proposes a technique to improve the intestinal colonization of bacteria by simply manipulating a single gene disruption, and it is expected to contribute to future research on the colonization of useful bacteria.

Acetaminophen-induced hepatotoxicity of cultured hepatocytes depends on timing of isolation from light-cycle controlled mice.

Most physiological changes follow a daily cycle in animals because their circadian rhythm is adjusted to and synchronized with sunlight. In particular, the circadian rhythm affects liver functions, including pharmacokinetics and metabolism. The influence of circadian rhythm has not been included in hepatotoxicity assays used in drug discovery and development. In this study, the contribution of circadian rhythm was investigated in acetaminophen-induced hepatotoxicity in mice and primary cultured hepatocytes. Hepatotoxicity was induced via the intraperitoneal administration of acetaminophen to a greater extent at night than during the day in mice. The sensitivity of acetaminophen-induced hepatotoxicity was consistent with the expression levels of acetaminophen-metabolizing enzyme and circadian genes. The host-derived circadian rhythm was still evident in the primary cultured hepatocytes within a day after their isolation from the liver. Primary cultured hepatocytes isolated at night were significantly more sensitive to acetaminophen than those isolated during the day. The sensitivity toward acetaminophen-induced hepatotoxicity depended on the circadian rhythm of the expression of acetaminophen-metabolizing genes and intracellular glutathione levels in primary cultured hepatocytes. These results obtained from cultured cells correspond to those in mice, suggesting that the timing of hepatocyte isolation is important when investigating drug metabolism and toxicity tests in culture.

Human iPS cell-derived astrocytes support efficient replication of progressive multifocal leukoencephalopathy-type JC polyomavirus.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system, in immunocompromised patients. Although PML used to be rare, recently the incidence of PML has risen due to an increase in immunosuppressive therapy. An in vitro JCPyV infection system could be used for anti-drug screening and investigation of tropism changes, but study of JCPyV in vitro has been limited due to the difficulty of efficiently propagating the virus in cultured cells. PML-type JCPyV efficiently propagates in primary human fetal and progenitor cell-derived astrocytes, but the preparation of cells from human fetuses is associated with severe ethical problems. In this study, human iPS cell-derived astrocytes were exposed to PML-type JCPyV. Infection, replication, and VP1 and T antigens of JCPyV were detected and confirmed in this culture. The non-coding control region (NCCR) of M1-IMRb was conserved in infected cells without point mutations. In addition, PML-type JCPyV genomic DNA in infected cells was detected as a single band of approximately 5.1 kbp, with no deletions. This is the first demonstration that human iPS cell-derived astrocytes efficiently support replication of PML-type JCPyV without production of defective interfering particles. These findings indicated that a culture system using human iPS cell-derived astrocyte would be useful for studies of PML, especially for screening anti-JCPyV drugs.

Acorbine, a Corbicula japonica-derived tripeptide containing non-proteinogenic amino acids, suppresses ethanol-induced liver injury.

Since ancient times, Corbicula extract has been believed in Japan to have hepatoprotective effects, but it remains unclear whether these claims are true, and if so, which component is responsible for hepatoprotection. In this study, we showed that Corbicula extract exerted a protective effect against liver damage. Recent work identified acorbine (ß-alanyl-ornithyl-ornithine), a novel tripeptide containing non-proteinogenic amino acids, in the extract of Corbicula japonica. Synthesized acorbine cured alcohol-induced liver damage in mice. In addition, acorbine purified from Corbicula extract exerted a protective effect against alcohol-induced hepatotoxicity in a culture liver model derived from mouse ES/iPS cells. Thus, acorbine is one of the components of Corbicula extract that protects hepatocytes against ethanol-induced death.

MazF activation causes ACA sequence-independent and selective alterations in RNA levels in Escherichia coli.

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.

Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8-cell embryo injection and aggregation.

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8-cell embryo aggregation, 8-cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8-cell embryo injection and 8-cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8-cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte-derived drug-metabolizing enzyme. Collectively, these findings indicate that the 8-cell embryo injection method is the most suitable for generation of PSC-derived organs via chimeric animal generation, particularly for the liver.

Transdifferentiation of mouse visceral yolk sac cells into parietal yolk sac cells in vitro.

The mouse embryonic yolk sac is an extraembryonic membrane that consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and functions in hematopoietic-circulation in the fetal stage. The present study was undertaken to examine the normal development of both murine VYS and PYS tissues using various molecular markers, and to establish a novel VYS cell culture system in vitro for analyzing differentiation potentials of VYS cells. RT-PCR and immunohistochemical analyses of gene expression in VYS and PYS tissues during development revealed several useful markers for their identification: HNF1ß, HNF4α, Cdh1 (E-cadherin), Krt8 and Krt18 for VYS epithelial cells, and Stra6, Snail1, Thbd and vimentin for PYS cells. PYS cells exhibited mesenchymal characteristics in gene expression and morphology. When VYS cells at 11.5 days of gestation were cultured in vitro for 7 days, the number of HNF1ß-, HNF4α-, E-cadherin- and cytokeratin-positive VYS epithelial cells was significantly reduced and, instead, Stra6-and vimentin-positive PYS-like cells increased with culture. RT-PCR analyses also demonstrated that gene expression of VYS markers decreased, whereas that of PYS markers increased in the primary culture of VYS cells. These data indicate that VYS epithelial cells rapidly transdifferentiate into PYS cells having mesenchymal characteristics in vitro, which may provide a culture system suitable for studying molecular mechanisms of VYS transdifferentiation into PYS cells and also epithelial-mesenchymal transition.

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ T cell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini.

The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.

In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor.

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

Acetaminophen-induced hepatotoxicity in a liver tissue model consisting of primary hepatocytes assembling around an endothelial cell network.

Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.

Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils.

BACKGROUND: Liver transplantation is a therapy for irreversible liver failure; however, at present, donor organs are in short supply. Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes. AIM: To investigate the possibility that hepatic progenitor cells (HPCs) prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine, we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells. METHODS: In vitro liver organoid tissue has been generated by accumulating collagen fibrils, fibroblasts, and HPCs on a mesh of polylactic acid fabric using a bioreactor; this was subsequently implanted into syngeneic wild-type mice. RESULTS: The in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy. CONCLUSION: Liver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system. This tissue was comparable to liver lobules, and with fibroblasts embedded in the network collagen fibrils of this artificial tissue, it is useful for reconstructing the hepatic interstitial structure.

Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides.

The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces.

OBJECTIVES: Osteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs). METHODS: Various cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro. RESULTS: Osteoclast formation was promoted on pillars (diameter, 1 µm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm). CONCLUSIONS: We demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 µm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.

Hepatocytes and Endothelial Networks in a Fluid-Based In Vitro Model of Liver Drug Metabolism.

Drug-induced liver toxicity remains a major cause of drug withdrawal from animal testing and human clinical trials. A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. Previously reported fluid-based systems have some advantages for hepatocyte culture, but have insufficient liver-specific functions because they simply involve moving conventional hepatocyte cultures from a dish into a fluid-based system. Importantly, these cultures have no liver tissue-specific structures that construct liver-specific cellular polarities, such as apical, basolateral, and basal faces. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions. Impact statement A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions.

Human DC3 Antigen Presenting Dendritic Cells From Induced Pluripotent Stem Cells.

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias toward erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells.

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

In vitro reprogramming of adult hepatocytes into insulin-producing cells without viral vectors.

The pancreas and the liver share the same endodermal origin. We have been studying whether mature hepatocytes can be induced to differentiate into pancreatic beta-cells by in vitro delivery of transcriptional factors using a non-viral approach. Here we showed that nucleofection allowed suitable transfection of primary hepatocytes employing various non-viral methods. We introduced either pancreatic and duodenal homeobox 1 (Pdx1) or neurogenin 3 (Ngn3), or both, into the mature cells using nucleofection. Co-expression of pdx1 and ngn3 using a bicistronic vector activated the transcription of various islet-related genes, and the transfected hepatocytes acquired the ability to synthesize and secrete insulin. Our results suggest that simultaneous expression of Pdx1 and Ngn3 is an excellent inducer of liver-to-pancreas reprogramming, and that reprogramming will occur even in mature somatic cells without the need for viral vectors. These findings are of considerable significance for further therapeutic development for various intractable diseases including diabetes.

Deficiency of tumour necrosis factor-alpha and interferon-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury.

AIMS: Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since it is now known that vascular injury involves an inflammatory response, we examined the role of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the neointimal formation after injury. METHODS AND RESULTS: Control (BALB/c), TNF-alpha-deficient (Tnf(-/-)), IFN-gamma-deficient (Ifng(-/-)), or double-deficient (Tnf(-/-)Ifng(-/-)) mice were subjected to wire-mediated vascular injury of the right femoral artery. Neointimal formation after injury was significantly reduced after the injury in the Tnf(-/-)Ifng(-/-) mice, compared to that in the control, Tnf(-/-), and Ifng(-/-) mice. Immunohistochemical analysis showed that TNF-alpha and IFN-gamma were expressed in neointimal lesions in the control mice, but not in mice with deficiency of the corresponding cytokine. No significant difference in re-endothelialization was observed among these groups. The number of proliferating cell nuclear antigen in the neointimal lesions was significantly decreased in the Tnf(-/-)Ifng(-/-) mice. Bone marrow transplantation experiments revealed that deficiency of TNF-alpha and IFN-gamma specifically in bone marrow cells significantly inhibited neointimal formation after vascular injury. CONCLUSION: The absence of TNF-alpha and IFN-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury, and thus, may provide new insights into the mechanisms underlying restenosis after PCI.