Induction of Krüppel-like factor 2 reduces K/BxN serum-induced arthritis.

Krüppel-like factor 2 (KLF2) critically regulates activation and function of monocyte, which plays important pathogenic role in progressive joint destruction in rheumatoid arthritis (RA). It is yet to be established the molecular basis of KLF2-mediated regulation of monocytes in RA pathogenesis. Herein, we show that a class of compound, HDAC inhibitors (HDACi) induced KLF2 expression in monocytes both in vitro and in vivo. KLF2 level was also elevated in tissues, such as bone marrow, spleen and thymus in mice after infusion of HDACi. Importantly, HDACi significantly reduced osteoclastic differentiation of monocytes with the up-regulation of KLF2 and concomitant down-regulation of matrixmetalloproteinases both in the expression level as well as in the protein level. In addition, HDACi reduced K/BxN serum-induced arthritic inflammation and joint destruction in mice in a dose-dependent manner. Finally, co-immunoprecipitation and overexpression studies confirmed that KLF2 directly interacts with HDAC4 molecule in cells. These findings provide mechanistic evidence of KLF2-mediated regulation of K/BxN serum-induced arthritic inflammation.

Role of Invasive Functional Assessment in Surgical Revascularization of Coronary Artery Disease.

In patients with stable coronary artery disease, percutaneous coronary intervention is associated with improved outcomes if the lesion is deemed significant by invasive functional assessment using fractional flow reserve. Recent studies have shown that a revascularization strategy using instantaneous wave-free ratio is noninferior to fractional flow reserve in patients with intermediate-grade stenoses. The decision to perform coronary artery bypass grafting surgery is usually based on anatomic assessment of stenosis severity by coronary angiography. The data on the role of invasive functional assessment in guiding surgical revascularization are limited. In this review, we discuss the diagnostic and prognostic significance of invasive functional assessment in patients considered for coronary artery bypass grafting. In addition, we critically discuss ongoing and future clinical trials on the role of invasive functional assessment in surgical revascularization.

Lost and found: Coronary stent retrieval and review of literature.

A 61-year-old man with a history of percutaneous coronary intervention (PCI) of the mid right coronary artery (RCA) with a drug eluting stent (DES), presented with non-ST segment elevation myocardial infarction. Coronary angiography demonstrated complex disease of the distal RCA as well as in-stent stenosis of the previously placed mid RCA stent. The patient underwent bifurcation PCI of the distal RCA followed by attempted intervention with a DES on the mid RCA lesion. The stent could not cross the lesion and eventually became dissociated from its delivery system. The lost stent was successfully retrieved using two different snaring systems. The procedure was terminated without further attempts for stent delivery. The patient had an uneventful recovery and underwent successful PCI of the mid RCA lesion one month later.

Enhanced peripheral dopamine impairs post-ischemic healing by suppressing angiotensin receptor type 1 expression in endothelial cells and inhibiting angiogenesis.

Increased circulating catecholamines have been linked with cardiovascular anomalies as well as with peripheral vascular diseases. Although the roles of epinephrine and norepinephrine have received considerable attention, the role of the other catecholamine, dopamine, has been less studied. Since dopamine is a potent endogenous inhibitor of angiogenesis and as angiogenesis is essential for ischemic healing, we therefore studied the role played by dopamine during ischemic healing using dopamine D2 receptor knockout (KOD2) mice. Although concentration of dopamine and its rate-limiting enzyme, tyrosine hydroxylase, was considerably high in the muscle tissues of wild-type and KOD2 mice with unilateral hind limb ischemia (HLI), recovery was significantly faster in the KOD2 mice compared to the wild-type controls, thereby indicating that peripheral dopamine might have a role in this healing process. In addition, we observed significant differences in post-ischemic angiogenesis between these two groups. Our study further revealed that elevated dopamine independently suppressed activation of local tissue-based renin-angiotensin system (RAS), a critical growth factor system stimulating angiogenesis in ischemia. Angiotensin II (ATII) and its receptor, angiotensin receptor type 1 (AT1R), are the key players in RAS-mediated angiogenesis. Dopamine acting through its D2 receptors in endothelial cells inhibited ATII-mediated angiogenesis by suppressing the expression of AT1R in these cells. This study thus for the first time demonstrates the role played by dopamine in prolonging post-ischemic recovery. Therefore, pharmacological intervention inhibiting the action of dopamine holds promise as future therapeutic strategy for the treatment of HLI and other peripheral arterial diseases.

Induction of ATM/ATR pathway combined with Vγ2Vδ2 T cells enhances cytotoxicity of ovarian cancer cells.

Many ovarian cancer cells express stress-related molecule MICA/B on their surface that is recognized by Vγ2Vδ2 T cells through their NKG2D receptor, which is transmitted to downstream stress-signaling pathway. However, it is yet to be established how Vγ2Vδ2 T cell-mediated recognition of MICA/B signal is transmitted to downstream stress-related molecules. Identifying targeted molecules would be critical to develop a better therapy for ovarian cancer cells. It is well established that ATM/ATR signal transduction pathways, which is modulated by DNA damage, replication stress, and oxidative stress play central role in stress signaling pathway regulating cell cycle checkpoint and apoptosis. We investigated whether ATM/ATR and its down stream molecules affect Vγ2Vδ2 T cell-mediated cytotoxicity. Herein, we show that ATM/ATR pathway is modulated in ovarian cancer cells in the presence of Vγ2Vδ2 T cells. Furthermore, downregulation of ATM pathway resulted downregulation of MICA, and reduced Vγ2Vδ2 T cell-mediated cytotoxicity. Alternately, stimulating ATM pathway enhanced expression of MICA, and sensitized ovarian cancer cells for cytotoxic lysis by Vγ2Vδ2 T cells. We further show that combining currently approved chemotherapeutic drugs, which induced ATM signal transduction, along with Vγ2Vδ2 T cells enhanced cytotoxicity of resistant ovarian cancer cells. These findings indicate that ATM/ATR pathway plays an important role in tumor recognition, and drugs promoting ATM signaling pathway might be considered as a combination therapy together with Vγ2Vδ2 T cells for effectively treating resistant ovarian cancer cells.

Two-year safety and effectiveness of the platinum chromium everolimus-eluting stent for the treatment of small vessels and longer lesions.

OBJECTIVES: To report 1- and 2-year clinical outcomes of patients receiving platinum chromium everolimus-eluting stents (PtCr-EES) in the prospective, single-arm PLATINUM small vessel (SV) and long lesion (LL) studies. BACKGROUND: Small vessel diameter and long lesion length are independently associated with increased risk of adverse cardiac events after drug-eluting stent implantation. METHODS: The PLATINUM SV study enrolled 94 patients with coronary artery lesions in vessels ≥2.25 mm to <2.50 mm in diameter and ≤28 mm in length. The PLATINUM LL study enrolled 102 patients with lesions >24 to ≤34 mm long in vessels ≥2.50 to ≤4.25 mm in diameter. The primary endpoint for both studies was target lesion failure (TLF) at 1 year compared to a prespecified performance goal based on outcomes with the TAXUS Express paclitaxel-eluting stent in small vessels and long lesions. RESULTS: One-year TLF rates with the PtCr-EES were significantly (P < 0.001) lower than the predetermined performance goals: 2.4% versus 21.1% in the SV cohort and 3.2% versus 19.4% in the LL cohort. Cumulative rates of TLF to 2 years were 4.7% in the SV cohort and 8.8% in the LL cohort. No myocardial infarction or ARC definite/probable stent thromboses occurred in either cohort through 2-year follow-up. CONCLUSIONS: The clinical efficacy and safety outcomes observed in these small vessel and long lesion cohorts support the use of the PtCr-EES in the treatment of small diameter vessels and long lesions.

Nanofiber-expanded human umbilical cord blood-derived CD34(+) cell therapy accelerates cutaneous wound closure in NOD/SCID mice.

Nanofiber-expanded human umbilical cord blood-derived CD34(+) cell therapy has been shown to have potential applications for peripheral and myocardial ischaemic diseases. However, the efficacies of expanded CD34(+) cell therapy for treating cutaneous wounds and its mechanisms of action have yet to be established. Using an excisional wound model in non-obese diabetic/severe combined immune deficient mice, we show herein that CD34(+) cells accelerate the wound-healing process by enhancing collagen synthesis, and increasing fibroblast cell migration within the wound bed. Concomitantly, reduced levels of matrix metalloproteinase (MMPs) such as MMP1, MMP3, MMP9 and MMP13 were detected in the wound beds of animals treated with CD34(+) cells compared with vehicle-treated controls. CD34(+) cells were found to mediate enhanced migration and proliferation of dermal fibroblast cells in vitro. Moreover, CD34(+) cells secrete collagen in a serum-deprived environment. In mechanistic studies, co-culture of CD34(+) cells with primary skin fibroblasts increased the expression of collagen1A1, a component of type 1 collagen, and decreased the expression of MMP1 in fibroblast cells in the presence of a proteasome inhibitor. Finally, CD34(+) cell-mediated functions were transcriptionally regulated by the c-Jun N-terminal kinases pathway. Collectively, these data provide evidence of therapeutic efficacy and a novel mechanism of nanofiber-expanded CD34(+) cell-mediated accelerated wound healing.

Innovative approach to preparing radial artery cocktails in response to manufacturer shortages of nitroglycerin and verapamil.

BACKGROUND: Transradial access has gained popularity over transfemoral access for cardiac catheterization, because of the decreased risk of bleeding, time to ambulation, and length of stay leading to improved patient satisfaction. One disadvantage of the radial artery approach is vasospasm, which can be prevented with the administration of verapamil and nitroglycerin in a pre- and postradial cocktail. Unfortunately, there have been manufacturer shortages for both of these medications. METHODS: The utilization of radial artery cocktails and other nitroglycerin compounding practices were evaluated in response to cost containment and waste reduction initiatives and to medication shortages. RESULTS: A modified process for supplying verapamil and nitroglycerin for the transradial approach via separate syringes enabled physicians to have quick access to the medications and to customize the cocktail based on the patient's needs. This process also decreased costs and minimized wastage. The change in practice decreased waste from 44% for preradial cocktail syringes and 66% for postradial cocktail syringes to 8.7%. DISCUSSION: This process for supplying the medications necessary to perform a radial artery catheterization and intracoronary nitroglycerin has allowed for conservation of commercial product supply.

Human Vγ2Vδ2 T cells limit breast cancer growth by modulating cell survival-, apoptosis-related molecules and microenvironment in tumors.

Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vδ2 T cell subset was shown to recognize and limit the growth of various tumors at various degrees. The differential recognition of the tumor cells by Vγ2Vδ2 T cells are yet to be defined. Our study reveals that γδ T cells limit in vitro growth of most breast tumor cells, such as SkBr7 (HER2+), MCF7 (ER+) and MDA-MB-231 (ER-) by inhibiting their survival and inducing apoptosis, except BrCa-MZ01 (PR+) cells. To investigate detail mechanisms of antineoplastic effects, we found that cell death was associated with the surface expression levels of MICA/B and ICAM1. Molecular signaling analysis demonstrated that inhibition of cell growth by γδ T cells was associated with the lower expression levels of cell survival-related molecules such as AKT, ERK and concomitant upregulation of apoptosis-related molecules, such as PARP, cleaved caspase 3 and tumor suppressor genes PTEN and P53. However, opposite molecular signaling was observed in the resistant cell line after coculture with γδ T cells. In vivo, antineoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the downregulation of survival signals, strong induction of apoptotic molecules, disruption of microvasculature and increased infiltration of tumor associated macrophages. These findings reveal that a complex molecular signaling is involved in γδ T cell-mediated antineoplastic effects.

Transgenic swine: expression of human CD39 protects against myocardial injury.

UNLABELLED: CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs. CONTROL: 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.

Recurrent Spontaneous Coronary Artery Dissection Presenting with STEMI, Once is Never Enough!

BACKGROUND: Spontaneous coronary artery dissection (SCAD) is an under-recognized cause of acute coronary syndrome in women. CASE PRESENTATION: We present a 40-year-old female who presented with recurrent STEMI due to recurrent SCAD 10 days after the index presentation in a different coronary territory. She was successfully treated both times with drug eluting stents. MRI showed an evidence of transmural infarction but her ejection fraction was only mildly reduced. She had good short term outcome. CONCLUSION: High index of suspicion is crucial for early diagnosis and prompt medical and interventional management in patients with SCAD.

Umbilical cord blood-selected CD133(+) cells exhibit vasculogenic functionality in vitro and in vivo.

BACKGROUND AIMS: Current clinical trials utilize non-selected bone marrow (BM) mononuclear cells (MNC) to augment vasculo genesis within ischemic vascular beds. Recent reports have identified a diminished number and function of hemat-opoietic stem cells (HSC) from aged and diseased patients. Umbilical cord blood (UCB) provides a potential robust allo-geneic source of HSC for therapeutic vasculogenesis. METHODS: MNC and magnetically isolated CD133(+) cells were assessed for viability (trypan blue) and surface phenotype (flow cytometry). To test in vivo functionality of the cells, NOD/SCID mice underwent ligation of the right femoral artery followed immediately by cell injection. Blood flow recovery, necrosis, BM engraftment of human cells and histologic capillary density were determined. Cells were tested for potential mechanisms mediating the in vivo effects, including migration, cytokine secretion and angiogenic augmentation (Matrigel assays). RESULTS: Surface expression analysis showed CD31 (PECAM) expression was greatly increased in UCB CD133(+) cells compared with BM MNC. At 28 days, perfusion ratios were highest in animals receiving UCB CD133(+) cells, while animals receiving BM CD133(+) cells and BM MNC demonstrated perfusion ratios statistically higher than in animals treated with cytokine media alone. Animals receiving CD133(+) cells showed a statistically higher capillary density, reduced severe digit necrosis and increased engraftment in the BM than animals treated with unselected BM MNC. In vitro studies showed equivalent migration to stromal-derived factor-1 (SDF-1), increased production of tumor necrosis factor alpha (TNF-alpha) and increased branch points with the co-incubation of CD133(+) cells with human umbilical vein endothelial cells (HUVEC) in the Matrigel angiogenesis assay. CONCLUSIONS: Taken together, UCB CD133(+) cells exhibit robust vasculogenic functionality compared with BM MNC in response to ischemia.

Ex vivo nanofiber expansion and genetic modification of human cord blood-derived progenitor/stem cells enhances vasculogenesis.

The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded approximately 225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF(164) and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

Myeloid Krüppel-Like Factor 2 Critically Regulates K/BxN Serum-Induced Arthritis.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease, and Krüppel-like factor 2 (KLF2) regulates immune cell activation and function. Herein, we show that in our experiments 50% global deficiency of KLF2 significantly elevated arthritic inflammation and pathogenesis, osteoclastic differentiation, matrix metalloproteinases (MMPs), and inflammatory cytokines in K/BxN serum-induced mice. The severities of RA pathogenesis, as well as the causative and resultant cellular and molecular factors, were further confirmed in monocyte-specific KLF2 deficient mice. In addition, induction of RA resulted in a decreased level of KLF2 in monocytes isolated from both mice and humans along with higher migration of activated monocytes to the RA sites in humans. Mechanistically, overexpression of KLF2 decreased the level of MMP9; conversely, knockdown of KLF2 increased MMP9 in monocytes along with enrichment of active histone marks and histone acetyltransferases on the MMP9 promoter region. These findings define the critical regulatory role of myeloid KLF2 in RA pathogenesis.

Nanofiber-expanded human CD34+ cells heal cutaneous wounds in streptozotocin-induced diabetic mice.

Despite advances in diabetic wound care, the significant number of amputations that occur every year demands more effective therapeutics. Herein, we offer an aminated polyethersulfone nanofiber-expanded human umbilical cord blood-derived CD34+ cells (henceforth CD34+ cells) effective therapy, tested in cutaneous wounds developed in streptozotocin-induced diabetic NOD/SCID mice. We show that systemic administration of CD34+ cells homed to the wound site and significantly accelerated wound closure. Wound closure was associated with improved re-epithelialization and increased neovascularization; and with decreased sustained pro-inflammatory activity of NF-κB and its downstream effector molecules TNF-α, IL-1ß, and IL-6 at the wound bed. This finding was further supported by the observation of a decreased number of myeloperoxidase positive neutrophils, and concomitantly increased levels of IL-10. In addition, improved granulation tissue formation was observed along with higher collagen deposition and myofibroblasts and decreased expressions of MMP-1. Mechanistically, CD34+ cells reduced the level of MMP-1 expression by inhibiting recruitment of NF-κB to the MMP-1 promoter site in dermal fibroblasts. In summary, we provide evidence of a novel nanofiber-expanded CD34+ stem cell therapeutic development for treating diabetic wounds by defining their cellular and molecular mechanisms.

Transvenous intramyocardial cellular delivery increases retention in comparison to intracoronary delivery in a porcine model of acute myocardial infarction.

BACKGROUND: Clinical trials using intracoronary (IC) delivery of cells have addressed efficacy but the optimal delivery technique is unknown. Our study aimed to determine whether transvenous intramyocardial (TVIM) approach was advantageous for cellular retention in AMI. METHODS: Domestic pigs (n = 4) underwent catheterization with coronary angiography and ventriculography prior to infarction and pre- and post-cells. Pigs underwent 90-minute balloon occlusion of the left anterior descending artery (LAD). After one week they were prepared for IC (n = 2) or TVIM (n = 2) delivery of bone marrow mononuclear cells (MNC) labeled with GFP. IC infusion used an over-the-wire catheter to engage the LAD and balloon inflation to prevent retrograde flow. Venography via the coronary sinus was used for TVIM delivery. The anterior interventricular vein was engaged with a guidewire allowing use of the TransAccess catheter that is outfitted with an ultrasound tip for visualization. Animals were sacrificed one hour after delivery and tissue was analyzed. RESULTS: Procedures were performed without complication and monitoring was uneventful. 1 x 10(8) MNC were isolated from each bone marrow (BM) preparation and 1 x 10(7) MNC delivered. Ventriculography at one week revealed wall motion abnormalities consistent with an anterior AMI. TVIM and IC delivery revealed mean 452 cells per section and 235 cells per section on average, respectively, in the infarct zone (P = 0.01). CONCLUSION: We have demonstrated that TVIM approach for cell delivery is feasible and safe. Moreover, this approach may provide an advantage over IC infusion in retention of the cellular product; however, larger studies will be necessary.

Long-Term Safety and Efficacy of Platinum Chromium Everolimus-Eluting Stents in Coronary Artery Disease: 5-Year Results From the PLATINUM Trial.

OBJECTIVES: The authors sought to evaluate the final 5-year safety and effectiveness of the platinum-chromium everolimus-eluting stent (PtCr-EES) in the randomized trial, as well as in 2 single-arm substudies that evaluated PtCr-EES in small vessels (diameter <2.5 mm; n = 94) and long lesions (24 to 34 mm; n = 102). BACKGROUND: In the multicenter, randomized PLATINUM (PLATINUM Clinical Trial to Assess the PROMUS Element Stent System for Treatment of De Novo Coronary Artery Lesions), the PtCr-EES was noninferior to the cobalt-chromium everolimus-eluting stent (CoCr-EES) at 1 year in 1,530 patients undergoing percutaneous coronary intervention. METHODS: Patients with 1 or 2 de novo coronary artery lesions (reference vessel diameter 2.50 to 4.25 mm, length ≤24 mm) were randomized 1:1 to PtCr-EES versus CoCr-EES. All patients in the substudies received PtCr-EES. The primary endpoint was target lesion failure (TLF), a composite of target vessel-related cardiac death, target vessel-related myocardial infarction, or ischemia-driven target lesion revascularization. RESULTS: In the randomized trial, the 5-year TLF rate was 9.1% for PtCr-EES and 9.3% for CoCr-EES (hazard ratio [HR]: 0.97; p = 0.87). Landmark analysis demonstrated similar TLF rates from discharge to 1 year (HR: 1.12; p = 0.70) and from 1 to 5 years (HR: 0.90; p = 0.63). There were no significant differences in the rates of cardiac death, myocardial infarction, target lesion or vessel revascularization, or stent thrombosis. PtCr-EES had 5-year TLF rates of 7.0% in small vessels and 13.6% in long lesions. CONCLUSIONS: PtCr-EES demonstrated comparable safety and effectiveness to CoCr-EES through 5 years of follow-up, with low rates of stent thrombosis and other adverse events. The 5-year event rates were also acceptable in patients with small vessels and long lesions treated with PtCr-EES. (The PLATINUM Clinical Trial to Assess the PROMUS Element Stent System for Treatment of De Novo Coronary Artery Lesions [PLATINUM]; NCT00823212; The PLATINUM Clinical Trial to Assess the PROMUS Element Stent System for Treatment of De Novo Coronary Artery Lesions in Small Vessels [PLATINUM SV]; NCT01498692; The PLATINUM Clinical Trial to Assess the PROMUS Element Stent System for Treatment of Long De Novo Coronary Artery Lesions [PLATINUM LL]; NCT01500434).

Generation of osteoporosis in immune-compromised mice for stem cell therapy.

To evaluate therapeutic efficacy and to investigate involved molecular mechanisms of cell-based therapy in osteoporosis, the generation of a clinically relevant model is critically important. Herein, we describe detailed methods in generation of an immune-deficient osteoporotic murine model, and application of human umbilical cord blood-derived stem cells to assess their therapeutic efficacy.