Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling.

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as Cys in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed PI3K-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K-Akt signaling by MVK reversed epidermal growth factor-induced negative regulation of autophagy and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary.

The female ovary contains a finite number of oocytes, and their release at ovulation becomes sporadic and disordered with aging and with obesity, leading to loss of fertility. Understanding the molecular defects underpinning this pathology is essential as age of childbearing and obesity rates increase globally. We identify that fibrosis within the ovarian stromal compartment is an underlying mechanism responsible for impaired oocyte release, which is initiated by mitochondrial dysfunction leading to diminished bioenergetics, oxidative damage, inflammation, and collagen deposition. Furthermore, antifibrosis drugs (pirfenidone and BGP-15) eliminate fibrotic collagen and restore ovulation in reproductively old and obese mice, in association with dampened M2 macrophage polarization and up-regulated MMP13 protease. This is the first evidence that ovarian fibrosis is reversible and indicates that drugs targeting mitochondrial metabolism may be a viable therapeutic strategy for women with metabolic disorders or advancing age to maintain ovarian function and extend fertility.

Intraovarian, Isoform-Specific Transcriptional Roles of Progesterone Receptor in Ovulation.

Progesterone receptor (PGR) activity is obligatory for mammalian ovulation; however, there is no established direct functional pathway explaining how progesterone receptor completely and specifically regulates oocyte release. This study examined the overarching cell- and isoform-specific effects of the PGR within each cellular compartment of the ovary, using mice null for the PGR (PRKO), as well as isoform-specific null mice. The PGR was expressed in ovarian granulosa and stromal cells and although PRKO ovaries showed no visible histological changes in preovulatory ovarian morphology, follicle rupture did not occur. Reciprocal ovarian transplant experiments established the necessity of ovarian PGR expression for ovulation. Cumulus-oocyte complexes of PRKO mice exhibited normal morphology but showed some altered gene expression. The examination of mitochondrial activity showed subtle differences in PRKO oocytes but no differences in granulosa cell respiration, glycolysis or ß-oxidation. Concurrently, RNA-seq identified novel functional pathways through which the PGR may regulate ovulation. PGR-A was the predominant transcriptionally active isoform in granulosa cells and 154 key PGR-dependent genes were identified, including a secondary network of transcription factors. In addition, the PGR regulated unique gene networks in the ovarian stroma. Collectively, we establish the effector pathways activated by the PGR across the ovarian cell types and conclude that PGR coordinates gene expression in the cumulus, granulosa and stromal cells at ovulation. Identifying these networks linking the PGR to ovulation provides novel targets for fertility therapeutics and nonhormonal contraceptive development.

Oligonucleotide correction of an intronic TIMMDC1 variant in cells of patients with severe neurodegenerative disorder.

TIMMDC1 encodes the Translocase of Inner Mitochondrial Membrane Domain-Containing protein 1 (TIMMDC1) subunit of complex I of the electron transport chain responsible for ATP production. We studied a consanguineous family with two affected children, now deceased, who presented with failure to thrive in the early postnatal period, poor feeding, hypotonia, peripheral neuropathy and drug-resistant epilepsy. Genome sequencing data revealed a known, deep intronic pathogenic variant TIMMDC1 c.597-1340A>G, also present in gnomAD (~1/5000 frequency), that enhances aberrant splicing. Using RNA and protein analysis we show almost complete loss of TIMMDC1 protein and compromised mitochondrial complex I function. We have designed and applied two different splice-switching antisense oligonucleotides (SSO) to restore normal TIMMDC1 mRNA processing and protein levels in patients' cells. Quantitative proteomics and real-time metabolic analysis of mitochondrial function on patient fibroblasts treated with SSOs showed restoration of complex I subunit abundance and function. SSO-mediated therapy of this inevitably fatal TIMMDC1 neurologic disorder is an attractive possibility.

Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation.

BACKGROUND: While molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses. Circulating tumor cells (CTCs) are released from primary and/or metastatic tumors into the peripheral blood. We examined whether CTCs have potential as biomarkers by checking the number of CTCs, as well as the BRAF genotype of individual CTCs, in melanoma patients undergoing BRAF/MEK inhibitor treatment. METHODS: CTCs were isolated from peripheral blood using a high-density dielectrophoretic microwell array, followed by labeling with melanoma-specific markers (MART-1 and/or gp100) and a leukocyte marker (CD45). The numbers of CTCs were analyzed in fifteen patients with stage 0-III melanoma. Furthermore, changes in CTC numbers were assessed in five patients with stage IV melanoma at four time points during BRAF/MEK inhibitor treatment, and the BRAF genotype was analyzed in CTCs isolated from one patient. RESULTS: We examined CTCs in patients with stage 0-III (five samples per stage: stage 0-I, stage II, and stage III), and detected CTCs even in patients with early disease (stage 0 and I). Interestingly, recurrence occurred in the lymph nodes of one stage I patient 2 years after the detection of a high number of CTCs in the patient's blood. The total number of CTCs in four of five patients with stage IV melanoma fluctuated in response to BRAF/MEK inhibitor treatment, suggesting that CTC number has potential for use as a drug response marker in advanced disease patients. Interestingly, one of those patients had CTCs harboring seven different BRAF genotypes, and the mutated CTCs disappeared upon BRAF/MEK inhibitor treatment, except for those harboring BRAFA598V. CONCLUSIONS: CTCs are present even in the early stage of melanoma, and the number of CTCs seems to reflect patients' responses to BRAF/MEK inhibitor treatment. Furthermore, genetic heterogeneity of BRAF may contribute to resistance to BRAF/MEK inhibitors. Our findings demonstrate the usefulness of CTC analysis for monitoring responses to targeted therapies in melanoma patients, and for understanding the mechanism of drug resistance.

Bioanalytical Method for the Determination of Hydroxyproline in Mouse Kidney by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection.

An analytical method was developed and validated for the measurement of hydroxyproline (Hyp) levels in mouse kidney by high-performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) using an analytical column specially designed for the LC/MS/MS analysis for intact amino acids. Tissue hydrolyzed with hydrochloric acid could be directly injected into the LC/MS/MS, as well as separated and detected using the deuterium labelled Hyp as an internal standard. The calibration curve showed good linearity from 5 to 500 nmol/mg of tissue; the precision and accuracy, including within- and between-run, were less than 6% and within 100 ± 6%, respectively.

C-Type Natriuretic Peptide Improves Left Ventricular Functional Performance at Rest and Restores Normal Exercise Responses after Heart Failure.

In heart failure (HF), the impaired left ventricular (LV) arterial coupling and diastolic dysfunction present at rest are exacerbated during exercise. C-type natriuretic peptide (CNP) is elevated in HF; however, its functional effects are unclear. We tested the hypotheses that CNP with vasodilating, natriuretic, and positive inotropic and lusitropic actions may prevent this abnormal exercise response after HF. We determined the effects of CNP (2 µg/kg plus 0.4 µg/kg per minute, i.v., 20 minutes) on plasma levels of cGMP before and after HF and assessed LV dynamics during exercise in 10 chronically instrumented dogs with pacing-induced HF. Compared with the levels before HF, CNP infusion caused significantly greater increases in cGMP levels after HF. After HF, at rest, CNP administration significantly reduced LV end-systolic pressure (PES), arterial elastance (EA), and end-diastolic pressure. The peak mitral flow (dV/dtmax) was also increased owing to decreased minimum LVP (LVPmin) and the time constant of LV relaxation (τ) (P < 0.05). In addition, LV contractility (EES) was increased. The LV-arterial coupling (EES/EA) was improved. The beneficial effects persisted during exercise. Compared with exercise in HF preparation, treatment with CNP caused significantly less important increases in PES but significantly decreased τ (34.2 vs. 42.6 ms) and minimum left ventricular pressure with further augmented dV/dtmax Both EES, EES/EA (0.87 vs. 0.32) were increased. LV mechanical efficiency improved from 0.38 to 0.57 (P < 0.05). After HF, exogenous CNP produces arterial vasodilatation and augments LV contraction, relaxation, diastolic filling, and LV arterial coupling, thus improving LV performance at rest and restoring normal exercise responses after HF.

High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%-90.0%) and excellent linear performance (R2 = 0.8189-0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Fatal case of multiple recurrences of endometrial stromal sarcoma after fertility-sparing management.

Several cases of the uterus being preserved after diagnosis of endometrial stromal sarcoma (ESS) have been reported. Most of these patients were alive and did not experience relapse, but this might have reflected the short follow-up period, given the indolent recurrence of ESS. We report the first fatal case of ESS 10 years after fertility-sparing management. A specimen obtained from the last operation showed loss of estrogen receptor status and expression of c-kit without c-kit or PDGFR-α gene mutations.

Overexpressed HIF-2α in Endothelial Cells Promotes Vascularization and Improves Random Pattern Skin Flap Survival.

BACKGROUND: The local skin flap procedure is very useful for reconstruction. However, flap necrosis caused by circulatory failure can occur at its distal portion. Hypoxia-inducible factors (HIFs) in endothelial cells (ECs) help to maintain ECs and promote vascularization, and HIF-2α is abundantly expressed in ECs. However, the mechanisms of action of HIF-2α in ECs are not yet fully understood. The aim of this study was to evaluate the in vivo effects of overexpression of HIF-2α in ECs on skin flap survival. METHODS: A random pattern skin flap (1.0 × 3.0 cm) was elevated on the dorsum of transgenic mice (Tg mice) with EC-specific HIF-2α conditional overexpression and wild-type littermate control mice (n = 6). Flap survival was evaluated on postoperative day 7. Tissue samples from the skin flaps were harvested and analyzed using Western blotting, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry. RESULTS: The HIF-2α mRNA and protein levels were significantly increased in the Tg mice when compared with control mice. Tg mice had significantly increased skin flap survival areas (72.0% ± 2.7%) when compared with wild-type mice (45.7% ± 1.1%). Moreover, histological examination revealed an increase in the subcutaneous blood vessel counts in the Tg mice. CONCLUSIONS: Specific overexpression of HIF-2α in ECs promoted vascularization and enhanced skin flap survival in vivo in a mouse model.

Limb salvage and vascular augmentation by microsurgical free flap transfer for treatment of neuropathic diabetic foot ulcers.

BACKGROUND: Neuropathic diabetic foot ulcers are generally deep and infectious wounds extending to the bone or joint. We performed microsurgical free flap transfer for limb salvage and vascular augmentation of feet with diabetic neuropathy. METHODS: Angiography was performed to identify any significant arterial disease. The free flaps were transplanted after resection of the damaged skin and infected bone. Flow-through or end-to-side anastomosis to the dorsalis pedis artery was performed to preserve the arterial blood flow to the residual foot. RESULTS: An anterolateral thigh flap and free flaps based on the subscapular artery system were transplanted in 1 and 10 patients, respectively. All flaps survived. Arterial flow on the distal side of the anastomosis was postoperatively confirmed in all patients. During a mean follow-up period of 52 months, the long-term complications observed were recurrent ulcers in 4 patients. The limb salvage rate was 100%, and 82% of patients achieved functional ambulation. CONCLUSIONS: Microsurgical flap transplantation is a safe and useful technique for minimal amputation of a diabetic neuropathic foot. Postoperative protection of the feet is important in order to avoid recurrence of foot ulceration. The use of protective footwear custom-tailored for each patient is strongly recommended.

ß3-Adrenergic receptor antagonist improves exercise performance in pacing-induced heart failure.

In heart failure (HF), the impaired left ventricular (LV) arterial coupling and diastolic dysfunction present at rest are exacerbated during exercise. We have previously shown that in HF at rest stimulation of ß3-adrenergic receptors by endogenous catecholamine depresses LV contraction and relaxation. ß3-Adrenergic receptors are activated at higher concentrations of catecholamine. Thus exercise may cause increased stimulation of cardiac ß3-adrenergic receptors and contribute to this abnormal response. We assessed the effect of L-748,337 (50 µg/kg iv), a selective ß3-adrenergic receptor antagonist (ß3-ANT), on LV dynamics during exercise in 12 chronically instrumented dogs with pacing-induced HF. Compared with HF at rest, exercise increased LV end-systolic pressure (PES), minimum LV pressure (LVPmin), and the time constant of LV relaxation (τ) with an upward shift of early diastolic portion of LV pressure-volume loop. LV contractility decreased and arterial elastance (EA) increased. LV arterial coupling (EES/EA) (0.40 vs. 0.51) was impaired. Compared with exercise in HF preparation, exercise after ß3-ANT caused similar increases in heart rate and PES but significantly decreased τ (34.9 vs. 38.3 ms) and LVPmin with a downward shift of the early diastolic portion of LV pressure-volume loop and further augmented dV/dtmax. Both EES and EES/EA (0.68 vs. 0.40) were increased. LV mechanical efficiency improved from 0.39 to 0.53. In conclusion, after HF, ß3-ANT improves LV diastolic filling; increases LV contractility, LV arterial coupling, and mechanical efficiency; and improves exercise performance.

Systemic preconditioning by a prolyl hydroxylase inhibitor promotes prevention of skin flap necrosis via HIF-1-induced bone marrow-derived cells.

BACKGROUND: Local skin flaps often present with flap necrosis caused by critical disruption of the blood supply. Although animal studies demonstrate enhanced angiogenesis in ischemic tissue, no strategy for clinical application of this phenomenon has yet been defined. Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in ischemic vascular responses, and its expression is induced by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We assessed whether preoperative stabilization of HIF-1 by systemic introduction of DMOG improves skin flap survival. METHODS AND RESULTS: Mice with ischemic skin flaps on the dorsum were treated intraperitoneally with DMOG 48 hr prior to surgery. The surviving area with neovascularization of the ischemic flaps was significantly greater in the DMOG-treated mice. Significantly fewer apoptotic cells were present in the ischemic flaps of DMOG-treated mice. Interestingly, marked increases in circulating endothelial progenitor cells (EPCs) and bone marrow proliferative progenitor cells were observed within 48 hr after DMOG treatment. Furthermore, heterozygous HIF-1α-deficient mice exhibited smaller surviving flap areas, fewer circulating EPCs, and larger numbers of apoptotic cells than did wild-type mice, while DMOG pretreatment of the mutant mice completely restored these parameters. Finally, reconstitution of wild-type mice with the heterozygous deficient bone marrow cells significantly decreased skin flap survival. CONCLUSION: We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells.

[Despite medication, overdrive pacing is required to stabilize the electrical storm associated with acute coronary syndrome: a case report].

A 75-year-old female complained of severe chest pain and was emergently admitted to our hospital because of anterior acute myocardial infarction. Emergent coronary angiography was performed and revealed occlusion in segment 7, so a stent was implanted. Lidocaine, carvedilol, amiodarone, magnesium, and nifekalant were administered successively because non-sustained ventricular tachycardia (NSVT) frequently appeared like an electrical storm. After nifekalant administration, QTc was significantly prolonged and torsades de pointes was induced. Overdrive pacing was performed and finally the NSVT was completely controlled. If fatal arrhythmias such as NSVT show resistance to medication, overdrive pacing should be considered to stabilize the arrhythmia associated with acute coronary syndrome.

[Coronary ectasia resulting in thrombotic coronary occlusion after warfarin interruption: a case report].

A 68-year-old man taking aspirin and warfarin for ectatic right coronary artery complained of chest pain and was admitted to our hospital with acute myocardial infarction. He had discontinued taking warfarin due to nail bleeding for a month. Coronary angiography revealed total occlusion at segment 3 of the ectatic right coronary artery with massive thrombus. Because of unsuccessful reperfusion by an aspiration device, a 5F straight catheter was inserted into the ectatic right coronary artery to aspirate the massive thrombus, and Thrombolysis in Myocardial Infarction grade 3 flow reperfusion was obtained. Intravascular ultrasonography showed "moyamoya" vessels in the ectatic right coronary artery, suggesting an abnormal coronary flow pattern, but there was no evidence of unstable plaque. Warfarization should be considered to treat ectatic coronary artery.

[Coronary artery embolism from ruptured plaque in the left main trunks with difficulty in detection of culprit lesion: a case report].

A 60-year-old man complained of severe chest pain and was emergently admitted to our hospital with a dignosis of anterior acute myocardial infarction. Emergent coronary angiography revealed significant stenosis in segment 7 and filling defect in segment 11 without flow delay. Haziness was observed in segment 5. Coronary thromboembolism was suspected, but the embolic source or culprit lesion was hard to detect. Intravascular ultrasonography detected ruptured plaque with lipid pooling in segment 5. Stent implantation for segment 5 was performed successfully and the patient had an excellent clinical course. Coronary thromboembolism is rare and intravascular ultrasonography may be useful to detect the culprit lesion.

Endogenous beta3-adrenoreceptor activation contributes to left ventricular and cardiomyocyte dysfunction in heart failure.

The objective of the present study was to test the hypothesis that endogenous beta(3)-adrenoreceptor (AR) activation contributes to left ventricular (LV) and cardiomyocyte dysfunction in heart failure (CHF). Stimulation of the beta(3)-AR inhibits cardiac contraction. In the failing myocardium, beta(3)-ARs are upregulated, suggesting that stimulation of beta(3)-ARs may contribute to depressed cardiac performance in CHF. We assessed the functional significance of endogenous beta(3)-AR activation in 10 conscious dogs before and after pacing-induced CHF. Under normal conditions, L-748,337, a specific beta(3)-AR antagonist, produced a mild increase in LV contractile performance assessed by the slope (E(es)) of the LV pressure-volume relation (18%, 6.2 +/- 0.9 vs. 7.3 +/- 1.2 mmHg/ml, P < 0.05) and the improved LV relaxation time constant (tau; 28.4 +/- 1.9 vs. 26.8 +/- 1.0 ms, P < 0.05). After CHF, the plasma norepinephrine concentration increased eightfold, and L-748,337 produced a larger increase in E(es) (34%, 3.8 +/- 0.7 vs. 5.1 +/- 0.8 mmHg/ml, P < 0.05) and a greater decrease in tau (46.4 +/- 4.2 vs. 41.0 +/- 3.9 ms, P < 0.05). Similar responses were observed in isolated myocytes harvested from LV biopsies before and after CHF. In the normal myocyte, L-748,337 did not cause significant changes in contraction or relengthening. In contrast, in CHF myocytes, L-748,337 produced significant increases in contraction (5.8 +/- 0.9 vs. 6.8 +/- 0.9%, P < 0.05) and relengthening (33.5 +/- 4.2 vs. 39.7 +/- 4.0 microm/s, P < 0.05). The L-748,337-induced myocyte response was associated with improved intracellular Ca(2+) concentration regulation. In CHF myocytes, nadolol caused a decrease in contraction and relengthening, and adding isoproterenol to nadolol caused a further depression of myocyte function. Stimulation of beta(3)-AR by endogenous catecholamine contributes to the depression of LV contraction and relaxation in CHF.

Diastolic mitral annular velocity during the development of heart failure.

OBJECTIVES: We sought to investigate the mechanism of reduced diastolic mitral annular velocity with diastolic dysfunction, despite elevated left atrial (LA) pressure. BACKGROUND: The peak rate of left ventricular (LV) early diastolic filling (E) and velocity of the mitral annulus due to long-axis lengthening (E(M)) are reduced in mild diastolic dysfunction. With more severe dysfunction, E increases in response to increased LA pressures. In contrast, E(M) decreases, despite increased LA pressure. METHODS: We studied eight dogs instrumented to measure LA pressure, LV pressure, and internal dimensions during the progressive development of heart failure (HF) produced by rapid pacing. RESULTS: Early diastolic filling decreased after four days of pacing from 114 +/- 32 to 88 +/- 22 ml/s (p < 0.05), but with more severe HF, it progressively increased to 155 +/- 32 ml/s (p < 0.05). In contrast, E(M) progressively decreased from 44 +/- 12 mm/s during the control period to 24 +/- 8 mm/s after four weeks (p < 0.05). Although E(M) was related to the time constant of LV relaxation (tau) (R(2) = 0.85), E was not. The latter occurred coincident with termination of the early diastolic LA to LV pressure gradient during all conditions. In contrast, with increasing HF, E(M) was progressively delayed after LA to LV pressure crossover by 37 +/- 12 ms (p < 0.05). The time from E to E(M) was related to tau (R(2) = 0.97). CONCLUSIONS: With slowed relaxation during the development of HF, E(M) is reduced and delayed so that it occurs after early, rapid filling. Thus, with slowed relaxation, E(M) does not respond to the early diastolic LA to LV pressure gradient, because it occurs when LV pressure is greater than or equal to LA pressure.