Sex differences and risk factors in recurrent ischemic stroke.

Introduction: Recurrent ischemic stroke (RIS) is associated with increased mortality and poor outcomes. Therefore, secondary prevention is critical for reducing the risk of recurrent stroke. Previous studies have found sex differences in risk factors in patients with first-ever stroke; however, the results have been inconsistent for recurrent stroke. Therefore, this study aimed to investigate whether there are significant sex differences in the clinical characteristics and risk factors for recurrent ischemic stroke. Methods: We retrospectively studied 787 patients with recurrent ischemic stroke after first-ever stroke confirmation using magnetic resonance imaging (MRI) after visiting a regional tertiary hospital between 2014 and 2020. Demographic characteristics, laboratory findings, and risk factors were compared between the male and female patients. In addition, multivariate logistic regression was performed to identify the independent factors associated with stroke recurrence in male patients. Results: Among the 787 patients, 466 (59.2%) were males. Males were younger than females (67.6 vs. 71.9 years). Females had higher rates of hypertension, diabetes mellitus, dyslipidemia, and overweight than those of males. However, the alcohol drinking and smoking rate were significantly higher in males than that in females. There were no statistically significant sex-based differences in the laboratory findings. Among males, hypertension, alcohol drinking, smoking and dyslipidemia was a significant risk factor for ischemic stroke recurrence. Conclusion: Hypertension and dyslipidemia were significant risk factors of recurrent ischemic stroke in both genders. Smoking and alcohol drinking were significant risk factors associated with ischemic stroke recurrence in males. Therefore, smoking cessation and alcohol abstinence are recommended after the first stroke to prevent recurrent ischemic stroke especially for males. Diabetes was a significant risk factor of ischemic stroke recurrence in females. More extensive studies are needed to understand the causal relationship of each factors with ischemic stroke recurrence according to sex differences and specification of preventive management is needed.

Mouse Pharmacokinetics and In Vitro Metabolism of SH-11037 and SH-11008, Synthetic Homoisoflavonoids for Retinal Neovascularization.

Cremastranone is a member of the homoisoflavanone family with anti-angiogenic activity in the eyes. SH-11037, a potent and selective synthetic homoisoflavonoid derived from cremastranone, was studied here for pharmacokinetics and metabolism characterization with a special focus on esterase-mediated hydrolysis. SH-11037 was shown to be converted rapidly and nearly completely to SH-11008 following an intravenous dose in mice. SH-11008 showed a high systemic clearance well exceeding the hepatic blood flow in mice. Neither SH-11037 nor SH-11008 were detected in plasma following oral administration of SH-11037 and SH-11008 in mice. Carboxylesterase was shown to be responsible for the rapid and quantitative hydrolysis of SH-11037 to SH-11008 in mouse plasma; the hydrolytic bioconversion was much slower in dog and human plasma, with butyrylcholinesterase and paraoxonase 1 likely being responsible. In vitro metabolism studies with liver S9 fractions suggested that SH-11008 was likely to have a high hepatic metabolic clearance with a predicted hepatic extraction ratio close to 1 in both mouse and human. In conclusion, SH-11037 and SH-11008 both appear to possess pharmacokinetic profiles suboptimal as a systemic agent. SH-11008 is suggested to possess a low potential for systemic toxicity suitable as a topical ocular therapeutic agent.

Small-molecule inhibitors of ferrochelatase are antiangiogenic agents.

Activity of the heme synthesis enzyme ferrochelatase (FECH) is implicated in multiple diseases. In particular, it is a mediator of neovascularization in the eye and thus an appealing therapeutic target for preventing blindness. However, no drug-like direct FECH inhibitors are known. Here, we set out to identify small-molecule inhibitors of FECH as potential therapeutic leads using a high-throughput screening approach to identify potent inhibitors of FECH activity. A structure-activity relationship study of a class of triazolopyrimidinone hits yielded drug-like FECH inhibitors. These compounds inhibit FECH in cells, bind the active site in cocrystal structures, and are antiangiogenic in multiple in vitro assays. One of these promising compounds was antiangiogenic in vivo in a mouse model of choroidal neovascularization. This foundational work may be the basis for new therapeutic agents to combat not only ocular neovascularization but also other diseases characterized by FECH activity.

Endolymphatic Hydrops in Patients With Vestibular Migraine and Concurrent Meniere's Disease.

Objective: Intravenous contrast agent enhanced, high-resolution magnetic resonance imaging of the inner ear (iMRI) confirmed that patients with Menière's disease (MD) and vestibular migraine (VM) could present with endolymphatic hydrops (EH). The present study aimed to investigate EH characteristics and their interrelation to neurotologic testing in patients with VM, MD, or VM with concurrent MD (VM-MD). Methods: Sixty-two patients (45 females, aged 23-81 years) with definite or probable VM (n = 25, 19 definite), MD (n = 29, 17 definite), or showing characteristics of both diseases (n = 8) were included in this study. Diagnostic workup included neurotologic assessments including video-oculography (VOG) during caloric stimulation and head-impulse test (HIT), ocular and cervical vestibular evoked myogenic potentials (o/cVEMP), pure tone audiometry (PTA), as well as iMRI. EH's degree was assessed visually and via volumetric quantification using a probabilistic atlas-based segmentation of the bony labyrinth and volumetric local thresholding (VOLT). Results: Although a relevant number of VM patients reported varying auditory symptoms (13 of 25, 52.0%), EH in VM was only observed twice. In contrast, EH in VM-MD was prevalent (2/8, 25%) and in MD frequent [23/29, 79.3%; χ2(2) = 29.1, p < 0.001, φ = 0.7]. Location and laterality of EH and neurophysiological testing classifications were highly associated (Fisher exact test, p < 0.005). In MD, visual semi-quantitative grading and volumetric quantification correlated highly to each other (r S = 0.8, p < 0.005, two-sided) and to side differences in VOG during caloric irrigation (vestibular EH ipsilateral: r S = 0.6, p < 0.05, two-sided). In VM, correlations were less pronounced. VM-MD assumed an intermediate position between VM and MD. Conclusion: Cochlear and vestibular hydrops can occur in MD and VM patients with auditory symptoms; this suggests inner ear damage irrespective of the diagnosis of MD or VM. The EH grades often correlated with auditory symptoms such as hearing impairment and tinnitus. Further research is required to uncover whether migraine is one causative factor of EH or whether EH in VM patients with auditory symptoms suggests an additional pathology due to MD.

A Knock-Down Cell-Based Study for the Functional Analysis of Chloride Intracellular Channel 1 (CLIC1): Integrated Proteomics and Microarray Study.

BACKGROUND: Previously, we detected that chloride intracellular channel 1 (CLIC1) was involved in the pathogenesis of atopic dermatitis (AD). OBJECTIVE: In this study, we aimed to use high-throughput screening (HTS) approaches to identify critical factors associated with the function of CLIC1 in knock-down cells. METHODS: We down-regulated CLIC1 in human A549 cells via siRNA and then conducted serial HTS studies, including proteomics integrated with a microarray and the implementation of bioinformatics algorithms. RESULTS: Together, these approaches identified several important proteins and genes associated with the function of CLIC1. These proteins and genes included tumor rejection antigen (gp96) 1, nucleophosmin, annexin I, keratin 1 and 10, FLNA protein, enolase 1, and metalloprotease 1, which were found using two-dimensional electrophoresis (2-DE) proteomics. Separately, NTNG1, SEMA5A, CLEC3A, GRPR, GNGT2, GRM5, GRM7, DNMT3B, CXCR5, CCL11, CD86, IL2, MNDA, TLR5, IL23R, DPP6, DLGAP1, CAT, GSTA1, GSTA2, GSTA5, CYP2E1, ADH1A, ESR1, ARRDC3, A1F1, CCL5, CASP8, DNTT, SQSTM1, PCYT1A, and SLCO4C1 were found using a DNA microarray integrated with PPI mapping. CONCLUSION: CCL11 is thought to be a particularly critical gene among the candidate genes detected in this study. By integrating the datasets and utilizing the strengths of HTS, we obtained new insights into the functional role of CLIC1, including the use of CLIC1-associated applications in the treatment of human diseases such as AD.

Functional study of acetaldehyde dehydrogenase 1 (ALDH1) in keratinocytes: microarray integrating bioinformatics approaches.

The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.Communicated by Ramaswamy H. Sarma.

Behavior of Muscle-Derived Stem Cells on Silica Nanostructured Substrates.

The aim of the present work was to evaluate the responses of rat muscle-derived stem cells (rMDSCs) to growth on silica nanostructured substrates (SN) with nanoscale topographic surfaces. SN of different sizes (SN-60, SN-150, SN-300, SN-500, and SN-700) were prepared using silica nanoparticles with sizes of 60-700 nm. The prepared SN showed roughness at the nanoscale level. The total number of adherent cells on SN increased with increasing nanoscale level and incubation time. The rMDSCs attached to SN-500 and SN-700 were extensively flattened, whereas those grown on SN-60, SN-150, and SN-300 were more rounded. The rank order of the cell length and height of attached rMDSCs at 5 d on different surfaces was SN-60 ≈ SN-150 >> SN-300 > SN-500 > SN-700 > glass. Compared with rMDSCs grown on SN-60, SN-150, or SN-300, those attached to SN-500 and SN-700 exhibited a distinct morphology with filopodial extensions and stronger expression of focal adhesion, integrin, and actin. An evaluation of the gene expression of adhered rMDSCs showed that rMDSCs grown on SN-300 exhibited a higher environmental stress response than those grown on glass or SN-700. Collectively, our data provide fundamental insight into the cellular response and gene expression of rMDSCs grown on nanostructured substrates.

Total Synthesis of Naturally Occurring 5,7,8-Trioxygenated Homoisoflavonoids.

Homoisoflavonoids are in the subclass of the larger family of flavonoids but have one more alkyl carbon than flavonoids. Among them, 5,7,8-trioxygenated homoisoflavonoids have not been extensively studied for synthesis and biological evaluation. Our current objective is to synthesize 2 5,7,8-trioxygenated chroman-4-ones and 12 5,7,8-trioxygenated homoisoflavonoids that have been isolated from the plants Bellevalia eigii, Drimiopsis maculata, Ledebouria graminifolia, Eucomis autumnalis, Eucomis punctata, Eucomis pallidiflora, Chionodoxa luciliae, Muscari comosum, and Dracaena cochinchinensis. For this purpose, 1,3,4,5-tetramethoxybenzene and 4'-benzyloxy-2',3'-dimethoxy-6'-hydroxyacetophenone were used as starting materials. Asymmetric transfer hydrogenation using Noyori's Ru catalyst provided 5,7,8-trioxygenated-3-benzylchroman-4-ones with R-configuration in high yield and enantiomeric excess. By selective deprotection of homoisoflavonoids using BCl3, the total synthesis of natural products including 10 first syntheses and three asymmetric syntheses has been completed, and three isomers of the reported dracaeconolide B could be provided. Our research on 5,7,8-trioxygenated homoisoflavonoids would be useful for the synthesis of related natural products and pharmacological applications.

Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches.

Previously, we detected that 14-3-3 protein epsilon (YWHAE) was involved in the pathogenesis of atopic dermatitis (AD) and tyrosinase-mediated pigmentation. In this study, we aimed to identify critical factors associated with YWHAE in human keratinocytes using high-throughput screening (HTS) approaches to reveal its functions in skin. We overexpressed YWHAE in human HaCaT keratinocytes and then conducted serial HTS studies, including RNA sequencing integrated with antibody arrays and the implementation of bioinformatics algorithms. Cumulatively, these approaches identified several novel genes in keratinocytes associated with the function of YWHAE including KRT9, KRT1, KRT6C, BST2, CIB2, APH1B, ACTC1, IFI27, TUBA1A, CAPN6, UTY, MX2, and MAPK15, based on RNA sequencing data, and MAPK1, MMP2, TYK2, NOS3, and CASP3, based on antibody array data. In particular, CD37 is a unique gene that was detected and validated in all the methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of YWHAE in skin keratinocytes. The approach used here could contribute to the clinical understanding of YWHAE-associated applications in the treatment of AD disease. AbbreviationsDAVIDthe database for annotation, visualization and integrated discoveryHTSHigh-throughput screeningKEGGKyoto Encyclopedia of Genes and GenomesPPIprotein-protein interactionsCommunicated by Ramaswamy H. Sarma.

Enantioselective Synthesis of Homoisoflavanones by Asymmetric Transfer Hydrogenation and Their Biological Evaluation for Antiangiogenic Activity.

Neovascular eye diseases are a major cause of blindness. Excessive angiogenesis is a feature of several conditions, including wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Development of novel antiangiogenic small molecules for the treatment of neovascular eye disease is essential to provide new therapeutic leads for these diseases. We have previously reported the therapeutic potential of anti-angiogenic homoisoflavanone derivatives with efficacy in retinal and choroidal neovascularization models, although these are racemic compounds due to the C3-stereogenic center in the molecules. This work presents asymmetric synthesis and structural determination of anti-angiogenic homoisoflavanones and pharmacological characterization of the stereoisomers. We describe an enantioselective synthesis of homoisoflavanones by virtue of ruthenium-catalyzed asymmetric transfer hydrogenation accompanying dynamic kinetic resolution, providing a basis for the further development of these compounds into novel experimental therapeutics for neovascular eye diseases.

An OMICS-based study of the role of C3dg in keratinocytes: RNA sequencing, antibody-chip array, and bioinformatics approaches.

Previously, we have identified the C3dg protein as an important player in the pathogenesis of atopic dermatitis (AD). In this study, we aimed to identify critical factors associated with C3dg in human keratinocytes based on high-throughput screening (HTS) approaches. We overexpressed C3dg in HaCaT human keratinocytes and conducted serial HTS studies, including RNA sequencing analysis integrated with antibody-chip arrays and implementation of bioinformatics algorithms (PPI mappings). Cumulatively, these approaches identified several novel C3dg-associated genes and proteins that are thought to be significantly involved in skin diseases including AD. These novel genes and proteins included LPA, PROZ, BLK, CLDN11, and FGF22, which are believed to play important roles in C3dg-associated skin functions in keratinocytes, as well as genes related to the two important pathways of systemic lupus erythematosus and Staphylococcus aureus infection. In particular, FGF22 is a unique gene that was detected and validated in all methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of C3dg in keratinocytes. The approach used here contributes to clinical understanding of C3dg-associated applications and may also be applicable to treatment of AD.

Mouse Pharmacokinetics and in Vitro Metabolism of (±)-Cremastranone.

The objective of this study was to characterize pharmacokinetics and metabolism of (±)-cremastranone (CMT) in mouse. Plasma concentrations of CMT following a single oral dose (10 mg/kg) were all below quantitation limit throughout 24-h time course, indicating poor oral bioavailability. Its plasma levels declined rapidly, with a half-life (t1/2) of 1.5 ± 0.3 min following a single intravenous dose (5 mg/kg). They were below the quantitation limit after 15 min post-dosing. CMT showed a high plasma clearance (CLp) of 7.73 ± 3.09 L/h/kg. Consistently, CMT was metabolized rapidly, with a t1/2 < 1 min when it was incubated with liver or intestine S9 fractions of mouse and human in the presence of cofactors for CYP450, uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT), and sulfotransferase (ST). Further studies showed that CMT was metabolized by CYP450, UGT, and ST in vitro in liver S9 fractions of mouse and human, with UGT being the major enzyme responsible for its rapid metabolism. CMT was metabolized by UGT and ST in intestine S9 fractions of mouse and human. Mono-demethylated (M1), mono-glucuronide (M2), and mono-sulfate (M3 and M4) metabolites were tentatively identified in vitro. In conclusion, the pharmacokinetics of CMT is suboptimal as a systemic agent, especially as an oral therapy, due to its extensive metabolism. This report provides possible structural modifications to design CMT derivatives with better pharmacokinetic properties.

Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization.

The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.

Ferrochelatase is a therapeutic target for ocular neovascularization.

Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.

Design, synthesis and biological evaluation of photoaffinity probes of antiangiogenic homoisoflavonoids.

A naturally occurring homoisoflavonoid, cremastranone (1) inhibited angiogenesis in vitro and in vivo. We developed an analogue SH-11037 (2) which is more potent than cremastranone in human retinal microvascular endothelial cells (HRECs) and blocks neovascularization in animal models. Despite their efficacy, the mechanism of these compounds is not yet fully known. In the course of building on a strong foundation of SAR and creating a novel chemical tool for target identification of homoisoflavonoid-binding proteins, various types of photoaffinity probes were designed and synthesized in which benzophenone and biotin were attached to homoisoflavanonoids using PEG linkers on either the C-3' or C-7 position. Notably, the photoaffinity probes linking on the phenol group of the C-3' position retain excellent activity of inhibiting retinal endothelial cell proliferation with up to 72nM of GI50.

A novel small molecule ameliorates ocular neovascularisation and synergises with anti-VEGF therapy.

Ocular neovascularisation underlies blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. These diseases cause irreversible vision loss, and provide a significant health and economic burden. Biologics targeting vascular endothelial growth factor (VEGF) are the major approach for treatment. However, up to 30% of patients are non-responsive to these drugs and they are associated with ocular and systemic side effects. Therefore, there is a need for small molecule ocular angiogenesis inhibitors to complement existing therapies. We examined the safety and therapeutic potential of SH-11037, a synthetic derivative of the antiangiogenic homoisoflavonoid cremastranone, in models of ocular neovascularisation. SH-11037 dose-dependently suppressed angiogenesis in the choroidal sprouting assay ex vivo and inhibited ocular developmental angiogenesis in zebrafish larvae. Additionally, intravitreal SH-11037 (1 µM) significantly reduced choroidal neovascularisation (CNV) lesion volume in the laser-induced CNV mouse model, comparable to an anti-VEGF antibody. Moreover, SH-11037 synergised with anti-VEGF treatments in vitro and in vivo. Up to 100 µM SH-11037 was not associated with signs of ocular toxicity and did not interfere with retinal function or pre-existing retinal vasculature. SH-11037 is thus a safe and effective treatment for murine ocular neovascularisation, worthy of further mechanistic and pharmacokinetic evaluation.

Synthesis and Biological Evaluation of Novel Homoisoflavonoids for Retinal Neovascularization.

Eye diseases characterized by excessive angiogenesis such as wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity are major causes of blindness. Cremastranone is an antiangiogenic, naturally occurring homoisoflavanone with efficacy in retinal and choroidal neovascularization models and antiproliferative selectivity for endothelial cells over other cell types. We undertook a cell-based structure-activity relationship study to develop more potent cremastranone analogues, with improved antiproliferative selectivity for retinal endothelial cells. Phenylalanyl-incorporated homoisoflavonoids showed improved activity and remarkable selectivity for retinal microvascular endothelial cells. A lead compound inhibited angiogenesis in vitro without inducing apoptosis and had efficacy in the oxygen-induced retinopathy model in vivo.

Evaluation of small intestine submucosa and poly(caprolactone-co-lactide) conduits for peripheral nerve regeneration.

The present study employed nerve guidance conduits (NGCs) only, which were made of small intestine submucosa (SIS) and poly(caprolactone-co-lactide) (PCLA) to promote nerve regeneration in a peripheral nerve injury (PNI) model with nerve defects of 15 mm. The SIS- and PCLA-NGCs were easily prepared by rolling of a SIS sheet and a bioplotter using PCLA, respectively. The prepared SIS- and PCLA-NGCs fulfilled the general requirement for use as artificial peripheral NGCs such as easy fabrication, reproducibility for mass production, suturability, sterilizability, wettability, and proper mechanical properties to resist collapsing when applied to in vivo implantation. The SIS- and PCLA-NGCs appeared to be well integrated into the host sciatic nerve without causing dislocations and serious inflammation. All NGCs stably maintained their NGC shape for 8 weeks without collapsing, which matched well with the nerve regeneration rate. Staining of the NGCs in the longitudinal direction showed that the regenerated nerves grew successfully from the SIS- and PCLA-NGCs through the sciatic nerve-injured gap and connected from the proximal to distal direction along the NGC axis. SIS-NGCs exhibited a higher nerve regeneration rate than PCLA-NGCs. Collectively, our results indicate that SIS- and PCLA-NGCs induced nerve regeneration in a PNI model, a finding that has significant implications in the future with regard to the feasibility of clinical nerve regeneration with SIS- and PCLA-NGCs prepared through an easy fabrication method using promising biomaterials.

The first synthesis of the antiangiogenic homoisoflavanone, cremastranone.

An antiangiogenic homoisoflavanone, cremastranone, was synthesized for the first time. This scalable synthesis, which includes selective demethylation, could be used to develop lead molecules to treat angiogenesis-induced eye diseases. Synthetic cremastranone inhibited the proliferation, migration and tube formation ability of human retinal microvascular endothelial cells, important steps in pathological angiogenesis.