Existence and uniqueness of neutral functional differential equations with sequential fractional operators.

In this research paper, we investigate the existence and uniqueness of solutions for neutral functional differential equations with sequential fractional orders, specifically involving the [Formula: see text]-Caputo operator. To obtain the desired results, we employ the Banach fixed point theorem (BFPT), a nonlinear variation of the Leray-Schauder fixed point theorem (SFPT), and the Krasnoselski fixed point theorem (KFPT). Additionally, we provide illustrative examples that demonstrate the key findings. Furthermore, we address a scenario where an initial value integral condition is considered.

Prognostic Role of BRAFV600E Cellular Localization in Melanoma.

BACKGROUND: Approximately half of cutaneous melanoma tissues harbor BRAFV600E mutations, resulting in a constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Nuclear-cytoplasmic transport machinery is dysregulated in neoplastic cells and alters the key regulatory proteins that can lead to tumor progression and drug resistance. The significance of nuclear localization of BRAFV600E has not been fully understood. We examined the clinical significance of intracellular localization of BRAFV600E in cutaneous melanoma. STUDY DESIGN: Immunohistochemical analysis of BRAFV600E was performed on formalin-fixed, paraffin-embedded specimens of cutaneous melanoma (n = 91). Staining intensity was graded in a blinded manner. Correlations to clinical factors were analyzed by Fisher's exact test and 2-tailed t-test. Localization of BRAFV600E was determined in melanoma cells, and we investigated their resistance to BRAFV600E-specific inhibitor according to nuclear localization in both in vitro and in vivo models. RESULTS: We included 91 patients, of whom 32% (29 of 91) had cytoplasmic BRAFV600E. Nuclear BRAFV600E was observed in 30% (27 of 91). Overall, BRAFV600E expression correlated with TNM stage (p = 0.011), mitotic activity (p = 0.010), and ulceration (p = 0.045). Nuclear BRAFV600E expression correlated with overall clinical stage (p < 0.001), tumor size (p < 0.001), regional lymph node (p < 0.017), depth of invasion (p = 0.005), Clark level (p < 0.001), mitotic activity (p < 0.001), ulceration (p < 0.001), and margin status (p = 0.017). On a cellular level, BRAFV600E was identified in the nucleus, and its translocation was serum dependent. Our in vitro and in vivo data revealed sequestration of BRAFV600E in the cytosol-sensitized resistant cells to vemurafenib; nuclear retention of BRAFV600E was associated with aggressiveness and drug resistance. CONCLUSIONS: Nuclear localization of BRAFV600E is associated with melanoma aggressiveness. Further multi-institutional studies are warranted to confirm the clinical relevance of nuclear localization of BRAFV600E.

ApoE deficiency promotes colon inflammation and enhances inflammatory potential oxidized-LDL and TNF-α in colon epithelial cells.

Although deficiency in Apolipoprotein E (ApoE) is linked to many diseases, its effect on colon homeostasis remains unknown.  ApoE appears to control inflammation by regulating NF-kB.  This study was designed to examine whether ApoE deficiency affects factors of colon integrity in vivo and given the likelihood that ApoE deficiency increases oxidized lipids and TNF-α, this study also examined whether such deficiency enhances the inflammatory potential of oxidized-LDL (oxLDL) and TNF-α, in colon epithelial cells in vitro   Here we show that ApoE deficiency is associated with chronic inflammation systemically and in colonic tissues as assessed by TNF-α levels.  Increased colon TNF-α mRNA coincided with a substantial increase in cyclooxygenase (COX)-2.  ApoE deficiency enhanced the potential of oxLDL and TNF-a to induce COX-2 expression as well as several other inflammatory factors in primary colon epithelial cells.   Interestingly, oxLDL enhanced TGF-ß expression only in ApoE-/-, but not in wild-type, epithelial cells.  ApoE deficiency appears to promote COX-2 expression enhancement through a mechanism that involves persistent NF-κB nuclear localization, PI3 and p38 MAP kinases but independently of Src.  In mice, ApoE deficiency promoted a moderate increase in crypt length, which was associated with opposing effects of an increase in cell proliferation and apoptosis at the bottom and top of the crypt, respectively.   : Our results support the notion that ApoE plays a central role in colon homeostasis and that ApoE deficiency may constitute a risk factor for colon pathologies.

Aldose reductase inhibition prevents allergic airway remodeling through PI3K/AKT/GSK3ß pathway in mice.

BACKGROUND: Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs). METHODS: Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFß1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling. RESULTS: In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFß1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFß1- induced activation of PI3K/AKT/GSK3ß pathway but not the phosphorylation of Smad2/3. CONCLUSION: Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFß1-induced Smad-independent and PI3K/AKT/GSK3ß-dependent pathway.

Aldose reductase inhibition suppresses the expression of Th2 cytokines and airway inflammation in ovalbumin-induced asthma in mice.

Airway inflammation induced by reactive oxygen species-mediated activation of redox-sensitive transcription factors is the hallmark of asthma, a prevalent chronic respiratory disease. In various cellular and animal models, we have recently demonstrated that, in response to multiple stimuli, aldose reductase (AR) regulates the inflammatory signals mediated by NF-kappaB. Because NF-kappaB-mediated inflammation is a major characteristic of asthma pathogenesis, we have investigated the effect of AR inhibition on NF-kappaB and various inflammatory markers in cellular and animal models of asthma using primary human small airway epithelial cells and OVA-sensitized/challenged C57BL/6 mice, respectively. We observed that pharmacological inhibition or genetic ablation of AR by small interfering RNA prevented TNF-alpha- as well as LPS-induced apoptosis; reactive oxygen species generation; synthesis of inflammatory markers IL-6, IL-8, and PGE(2); and activation of NF-kappaB and AP-1 in small airway epithelial cells. In OVA-challenged mice, we observed that administration of an AR inhibitor markedly reduced airway hyperresponsiveness, IgE levels, eisonophils infiltration, and release of Th2 type cytokines in the airway. Our results indicate that AR inhibitors may offer a novel therapeutic approach to treat inflammatory airway diseases such as asthma.

Inhibition of aldose reductase prevents experimental allergic airway inflammation in mice.

BACKGROUND: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress-induced inflammation by affecting the NF-kappaB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. METHODS AND FINDINGS: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-kappaB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E(2), IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice. CONCLUSIONS: These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-kappaB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.

Role of advanced glycation end products with oxidative stress in resistance artery dysfunction in type 2 diabetic mice.

OBJECTIVE: Type 2 diabetes is associated with increased advanced glycation end product (AGE) formation and vasculopathy. We hypothesized that AGEs contribute to resistance artery dysfunction. METHODS AND RESULTS: Type 2 diabetic db(-)/db(-) (diabetic) and nondiabetic db(-)/db(+) (control) mice were treated with the AGE inhibitor (aminoguanidine: 50 mg/Kg/d) for 3 months. Isolated mesenteric resistance arteries (MRAs) were mounted in an arteriograph. Pressure-induced myogenic tone (MT) was increased in diabetic mice but was unaffected by aminoguanidine treatment. Phenylephrine-induced contraction and nitric oxide donor-induced endothelium-independent relaxation were similar in all groups. In diabetic mice, endothelium-dependent relaxation in response to shear-stress or acetylcholine was altered and was associated with reduced eNOS protein and mRNA expression. Aminoguanidine treatment improved endothelial function and restored eNOS expression. AGE formation and hypoxia markers (plasminogen activator inhibitor 1 and Bnip3) were increased in MRA from diabetic mice and normalized with Aminoguanidine. Primary cultured endothelial cells (ECs) isolated from resistance arteries subjected to high glucose for 48 hours showed decreased eNOS expression and phosphorylation in response to calcium ionophore. High glucose decreased antioxidant protein (MnSOD) and increased prooxidant proteins (gp91phox) expression leading to increased oxidative stress generation, as assessed by DHE staining and endothelial NADH/NADPH oxidase activity. The preincubation of ECs with aminoguanidine restored eNOS-phosphorylation and expression as well as the balance between pro- and antioxidant factors induced by high glucose. CONCLUSIONS: We provide evidence of a link between AGEs, oxidative stress, and resistance artery EC dysfunction in type 2 diabetic mice. Thus, AGEs and oxidative stress may be a potential target for overcoming diabetic microvessels complications.

Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, alphavbeta3-integrin, and TGF-beta1 in response to ANG II and high glucose.

This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, alphavbeta(3)-integrin, and transforming growth factor (TGF)-beta1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-beta1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-beta1, alphavbeta(3)-integrin, and collagen type 1 release in the conditioned media. TGF-beta1 bioactivity and Smad2 phosphorylation were alphavbeta(3)-integrin-dependent, since beta(3)-integrin antibody and alphavbeta(3)-integrin inhibitor (SB-223245, 10 microM) significantly prevented TGF-beta1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 microM) reduced alphavbeta(3)-integrin, TGF-beta1, and collagen type 1 content. Additionally, alphavbeta(3)-integrin antibody, SB-223245, TGF-beta1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, alphavbeta(3)-integrin, and TGF-beta1 signaling. This pathway could be a potential target for overcoming small artery complications in diabetes and hypertension.

Elevated epidermal growth factor receptor phosphorylation induces resistance artery dysfunction in diabetic db/db mice.

OBJECTIVE: We previously showed epidermal growth factor receptor (EGFR) transactivation to be key mechanism in the regulation of resistance artery myogenic tone. Type 2 diabetes is associated with microvascular complications. We hypothesized that elevated EGFR phosphorylation contributes to resistance artery dysfunction in type 2 diabetes. RESEARCH DESIGN AND METHODS AND RESULTS: Diabetic db/db and nondiabetic (control) mice were treated with EGFR inhibitor (AG1478; 10 mg x kg(-1) x day(-1)) for 2 weeks. Isolated coronary artery and mesenteric resistance artery (MRA) were mounted in an arteriograph. Pressure-induced myogenic tone was increased in MRA and coronary artery from diabetic mice and normalized by AG1478. Phenylephrine-induced contraction and nitric oxide donor-induced relaxation were similar in all groups. Endothelium-dependent relaxation in response to shear stress and acetylcholine of MRA and coronary artery from diabetic mice was altered and associated with reduced endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Treated diabetic mice with AG1478 improved coronary artery and MRA endothelial function and restored eNOS expression. Immunostaining and Western blot analysis showed increased endothelial and smooth muscle cell EGFR phosphorylation of MRA and coronary artery from diabetic mouse, which was reduced by AG1478. Primary cultured endothelial cells from resistance arteries treated with high glucose for 48 h showed an increase of EGFR phosphorylation associated with eNOS expression and phosphorylation decrease in response to calcium ionophore. Pretreatment of endothelial cells with AG1478 prevented the effect of high glucose. CONCLUSIONS: This study provides evidence of the role of elevated EGFR phosphorylation in coronary artery and MRA dysfunction in diabetic db/db mice. Therefore, EGFR should be a potential target for overcoming diabetic small artery complications.

Role of ACE/AT2R complex in the control of mesenteric resistance artery contraction induced by ACE/AT1R complex activation in response to Ang I.

OBJECTIVES: In this study, we will determine the function of the interaction between AT2R and ACE, and AT1R and ACE in the control of mesenteric resistance artery (MRA) tone from normotensive (NT) and Angiotensin II (AII)-dependent hypertensive (HT) mice. METHODS-RESULTS: Hypertension was induced by infusion of Ang-II (200 ng/kg/day) for 3 weeks. Freshly MRA (100-120 microm) were isolated from HT and NT mice and mounted in an arteriograph. Dose-response of Ang-I induced a similar contraction of MRA from NT and HT mice, which was increased after endothelium removal. AT2R antagonist (PD123319, 1 microM) significantly increased Ang-I-induced contraction of MRA from NT but not from HT mice. In addition, PD123319 significantly increased in vivo blood pressure in response to Ang-I. Luminal incubation with ACE-antibody (50 ng/ml) to block only endothelial ACE function significantly enhanced Ang-I-induced contraction of MRA from NT mice. ACE inhibitor (captopril, 10 microM) completely blocked Ang-I-induced contraction of MRA from both animals and prevented the increased blood pressure. Freshly isolated MRA subjected to immunoprecipitation, Western blot analysis and RT-PCR revealed AT1R/ACE and AT2R/ACE complexes formation, and similar AT1R, AT2R, and ACE expression level in both groups. CONCLUSION: The present findings show the existence of ACE/AT2R and ACE/AT1R complexes on endothelial cells and VSMC, respectively. ACE/AT2R complex plays a modulator effect on ACE/AT1R-SMC-induced contraction of MRA, which is altered in hypertension.

Poly(ADP-ribose) polymerase-1 inhibition prevents eosinophil recruitment by modulating Th2 cytokines in a murine model of allergic airway inflammation: a potential specific effect on IL-5.

We recently used a murine model of allergic airway inflammation to show that poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in the pathogenesis of asthma-related lung inflammation. In this study, we show that PARP-1 inhibition, by a novel inhibitor (TIQ-A) or by gene deletion, prevented eosinophilic infiltration into the airways of OVA-challenged mice. Such impairment of eosinophil recruitment appeared to take place after IgE production. OVA challenge of wild-type mice resulted in a significant increase in IL-4, IL-5, IL-10, IL-13, and GM-CSF secretions. Although IL-4 production was moderately affected in OVA-challenged PARP-1(-/-) mice, the production of IL-5, IL-10, IL-13, and GM-CSF was completely inhibited in ex vivo OVA-challenged lung cells derived from these animals. A single TIQ-A injection before OVA challenge in wild-type mice mimicked the latter effects. The marked effect PARP-1 inhibition exerted on mucus production corroborated the effects observed on the Th2 response. Although PARP-1 inhibition by gene knockout increased the production of the Th1 cytokines IL-2 and IL-12, the inhibition by TIQ-A exerted no effect on these two cytokines. The failure of lung cells derived from OVA-challenged PARP-1(-/-) mice to synthesize GM-CSF, a key cytokine in eosinophil recruitment, was reestablished by replenishment of IL-5. Furthermore, intranasal administration of IL-5 restored the impairment of eosinophil recruitment and mucus production in OVA-challenged PARP-1(-/-) mice. The replenishment of either IL-4 or IgE, however, did not result in such phenotype reversals. Altogether, these results suggest that PARP-1 plays a critical role in eosinophil recruitment by specifically regulating the cascade leading to IL-5 production.

Correlation between decreased sensitivity of the Daudi lymphoma cells to VP-16-induced apoptosis and deficiency in DNAS1L3 expression.

A functional relationship between the apoptotic endonuclease DNAS1L3 and the chemotherapeutic drug VP-16 was established. The lymphoma cell line, Daudi, exhibited a significant resistance to VP-16 treatment in comparison to the lymphoma/leukemia cell line, U-937. While U-937 cells degraded their DNA into internucleosomal fragments, Daudi cells failed to undergo such fragmentation in response to the drug. Activation of both caspase-3 and DNA fragmentation factor was not sufficient to trigger internucleosomal DNA fragmentation in Daudi cells. No correlation was found between expression levels of topoisomerase-II, Pgp, Bcl-2, Bax, or Bad and decreased sensitivity of Daudi cells to VP-16. Daudi cells failed to express DNAS1L3 and ectopic expression of this protein significantly sensitized the cells to VP-16. An enhancement of caspase-3 activity and collapse of mitochondrial membrane potential underlie DNAS1L3-mediated sensitization of Daudi cells to VP-16, which may be a direct result of DNAS1L3-mediated increase in PARP-1-activating DNA breaks after VP-16 treatment. Our results suggest that DNAS1L3 plays an active role in lymphoma cell sensitization to VP-16 and that its deficiency may constitute a novel mechanism of drug resistance in these cells.