Adipokine-Cytokine Profile in Patients with Unstable Atherosclerotic Plaques and Abdominal Obesity.

The goal of the research was to study the levels of adipokines and their associations with unstable atherosclerotic plaques in patients with coronary atherosclerosis and abdominal obesity (AO). METHODS: The study included 145 men aged 38-79 with atherosclerosis of the coronary arteries (CA) and stable angina pectoris II-III FC who were hospitalized for coronary bypass surgery (2011-2022). The final analysis included 116 patients. Notably, 70 men had stable plaques in the CA (of which 44.3% had AO), and 46 men had unstable plaques in the CA (of which 43.5% had AO). Adipocytokine levels were determined using multiplex analysis (Human Metabolic Hormone V3 panel). RESULTS: In the subgroup of patients with unstable plaques, patients with AO had a GLP-1 level that was 1.5 times higher and a lipocalin-2 level that was 2.1 times lower, respectively. GLP-1 is direct, and lipocalin-2 is inversely associated with AO in patients with unstable plaques. Among patients with AO, the level of lipocalin-2 in patients with unstable plaques was 2.2 times lower than in patients with stable plaques in the CA. The level of lipocalin-2 was inversely associated with the presence of unstable atherosclerotic plaques in the CA. CONCLUSION: GLP-1 is directly associated with AO in patients with unstable atherosclerotic plaques. Lipocalin-2 is inversely associated with unstable atherosclerotic plaques in patients with AO.

Deep Hypothermic Circulatory Arrest vs. Antegrade Cerebral Perfusion in Cerebral Protection during the Surgical Treatment of Chronic Dissection of the Ascending and Arch Aorta.

Circulatory arrest during aortic surgery presents a risk of neurological complications. The present study aimed to investigate the effectiveness of deep hypothermic circulatory arrest (DHCA) vs. antegrade cerebral perfusion (ACP) in cerebral protection during the surgical treatment of chronic dissection of the ascending and arch aorta and to assess the quality-of-life (QoL) in the long-term postoperative period with respect to the used cerebral protection method. In a prospective, randomized study, 58 patients with chronic type I aortic dissection who underwent ascending aorta and aortic arch replacement surgery were included. Patients were allocated in two groups: 29 patients who underwent surgery under moderate hypothermia (24°C) combined with ACP and 29 patients who underwent surgery under DHCA (18°C) with craniocerebral hypothermia. The regional hemoglobin oxygen saturation (rSO2, %) were compared during surgery, neurological complications were analyzed during the early postoperative period, QoL was compared in the long-term postoperative period (1-year follow-up). During the early postoperative period, 37.9% of patients in the DHCA group exhibited neurological complications, compared with 13.8% of those in the ACP group (p < .05). The risk of neurological complications in the early postoperative period was dependent on the extent of rSO2 decrease during circulatory arrest. In the ACP group, rSO2 decreased by ≤17% from baseline during circulatory arrest. In the DHCA group, a more profound decrease in rSO2 (>30%) was recorded (p < .05). QoL in the long-term period after surgery improved, but it was not dependent on the cerebral protection method used during surgery. ACP during aortic replacement demonstrated the most advanced properties of cerebral protection that can be evidenced by a lesser degree of neurological complications, compared with patients who underwent surgery under conditions of DHCA. QoL after surgery was not dependent on the cerebral protection method used during surgery.

Minimally invasive versus conventional methods for aortic root surgery: Choosing the right approach.

OBJECTIVE: Partial upper sternotomy is preferred for isolated aortic valve replacement because of its optimal surgical visibility and favorable cosmetic outcomes; however, it is not commonly used for aortic root surgery, and the conventional median sternotomy is still the preferred method for most surgeons. We aimed to compare the safety and effectiveness of a minimally invasive approach (partial sternotomy [PS]) and conventional approach (median sternotomy [FS]) for aortic root surgery. METHODS: Patients who underwent aortic root surgery at our hospital from 2016 to 2021 were retrospectively enrolled and divided into two groups. After propensity score matching, the conventional group included 156 patients and the minimally invasive group-57 patients. RESULTS: Bicuspid aortic valves were observed in 63 (40.4%) and 33 (57.9%) patients in the FS and PS groups, respectively. Valve-sparing surgery was performed on 69 (44.2%) and 30 (52.6%) patients in the FS and PS groups, respectively. The minimally invasive approach was beneficial in terms of blood loss during the first 24 h after surgery (p = 0.029) and postoperative blood transfusion (p = 0.023). The survival rates and freedom from reoperation or severe aortic regurgitation after the David procedure were comparable between the standard and minimally invasive groups (p = 0.25; p = 0.66) at mid-term follow-up. CONCLUSIONS: A minimally invasive approach for aortic root surgery can be safely performed as the standard approach. Partial upper sternotomy has the advantage of lower blood loss in the early postoperative period and does not negatively affect the results of valve-sparing root replacement.

Diagnosis and Treatment Patterns of Chronic Thromboembolic Pulmonary Hypertension in Russia, Kazakhstan, Turkey, Lebanon, and Saudi Arabia: A Registry Study.

BACKGROUND: Patients with chronic thromboembolic pulmonary hypertension (CTEPH) in countries with limited resources have, to date, been poorly represented in registries. OBJECTIVE: This work assesses the epidemiology, diagnosis, hemodynamic and functional parameters, and treatment of CTEPH in Russia, Kazakhstan, Turkey, Lebanon, and Saudi Arabia. METHODS: A prospective, cohort, phase IV, observational registry with 3-year follow-up (n = 212) in patients aged ≥ 18 years diagnosed with CTEPH was created. Clinical, hemodynamic, and functional parameters were obtained at an initial visit, follow-up visits, and a final visit at the end of 3 years' observation or end of follow-up. Data were recorded on electronic case report forms. Parameters evaluated included 6-minute walking distance (6MWD), use of pulmonary endarterectomy (PEA), balloon pulmonary angioplasty (BPA), pulmonary hypertension (PH)-targeted therapy, and survival. All statistical analyses were exploratory and descriptive, and were performed in the overall population. RESULTS: The most common symptoms were typical of those expected for CTEPH. Almost 90% of patients underwent right heart catheterization at diagnosis or initial study visit. In total, 66 patients (31%) underwent PEA before the initial visit; 95 patients (45%) were considered operable, 115 (54%) were inoperable, and two (1%) had no operability data. Only 26 patients (12%) had been assessed for BPA at their initial visit. PH-targeted therapy was documented at diagnosis for 77 patients (36%), most commonly a phosphodiesterase type 5 inhibitor (23%). Use of PH-targeted therapy increased to 142 patients (67%) at the initial visit, remaining similar after 3 years. Use of riociguat increased from 6% of patients at diagnosis to 38% at 3 years. Between baseline and end of observation, results for patients with paired data showed an increase in 6MWD. Survival at the end of observation was 88%. CONCLUSIONS: These data highlight the current diagnosis and management of CTEPH in the participating countries. They show that early CTEPH diagnosis remains challenging, and use of off-label PH-targeted therapy is common. CLINICALTRIALS: gov: NCT02637050; registered December 2015.

Cytokine-induced alterations of α7 nicotinic receptor in colonic CD4 T cells mediate dichotomous response to nicotine in murine models of Th1/Th17- versus Th2-mediated colitis.

Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4(+)CD62L(+) T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory α7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in regulating colonic inflammation.

Redo aortic root surgery post-Ross procedure.

Background: Despite numerous advantages of the Ross procedure, it presents a risk of late autograft and right ventricular outflow tract conduit failure. This study aimed to analyze the outcomes of autograft dysfunction reoperations using autograft-sparing and root replacement techniques. Methods: Between 2015 and 2023, 49 patients underwent redo root surgery in our institution. Autograft valve-sparing procedures (VSP) were performed in 20 cases and the Bentall procedure (BP) in 29 patients. The short and long-term clinical outcomes along with echocardiographic results of VSP and BP were investigated. Results: Overall early mortality rate was 2.0% with no significant difference between the groups. Severe autograft valve insufficiency at the time of redo (OR 4.07, P = 0.03) and patient age (OR 1.07, P = 0.04) were associated with a valve replacement procedure instead of VSP. The median follow-up duration was 34 months. No late deaths occurred in either group. Freedom from VSP failure and aortic prosthesis dysfunction were 93.8% and 94.1% in the VSP and BP groups, respectively. No reoperations were necessary in either group. Conclusion: Redo aortic root surgery can be safely performed in patients with autograft failure. Both root replacement and autograft valve-sparing procedures demonstrated acceptable results at mid-term follow-up. Early redo surgery pre-empting severe aortic insufficiency increases the likelihood of preservation of the dilated autograft valve.

Novel cholinergic peptides SLURP-1 and -2 regulate epithelialization of cutaneous and oral wounds.

It is well established that auto/paracrine acetylcholine (ACh) is essential for wound epithelialization, and that the mechanisms include regulation of keratinocyte motility and adhesion via nicotinic ACh receptors (nAChRs). Keratinocyte nAChRs can be also activated by non-canonical ligands, such as secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP)-1 and -2. In this study, we determined effects of recombinant (r)SLURP-1 and-2 on migration of human epidermal and oral keratinocytes under agarose and epithelialization of cutaneous and oral mucosal excisional wounds in mice, and also identified nAChRs mediating SLURP signals. Both in vitro and in vivo, rSLURP-1 decreased and SLURP-2 increased epithelialization rate. The mixture of both peptides accelerated epithelialization even further, indicating that their simultaneous signaling renders an additive physiologic response. The specificity of rSLURP actions was illustrated by similar effects on cutaneous and oral wounds, which feature distinct responses to injury, and also by abrogation of rSLURP effects with neutralizing antibodies. rSLURP-1 acted predominantly via the α7 nAChR-coupled up-regulation of the sedentary integrins α2 and α3 , whereas SLURP-2--through α3, and α9 nAChRs up-regulating migratory integrins α5 and αV . The biologic effects of rSLURPs required the presence of endogenous ACh, indicating that auto/paracrine SLURPs provide for a fine tuning of the physiologic regulation of crawling locomotion via the keratinocyte ACh axis. Since nAChRs have been shown to regulate SLURP production, cholinergic regulation of keratinocyte migration appears to be mediated by a reciprocally arranged network. The cholinergic peptides, therefore, may become prototype drugs for the treatment of wounds that fail to heal.

Results of concomitant cryoablation for atrial fibrillation during mitral valve surgery.

OBJECTIVES: Concomitant atrial fibrillation ablation during mitral valve (MV) surgery using radio frequency energy sources has been reported previously with excellent outcomes. However, data regarding the effectiveness of concomitant cryoablation remain limited. This study aimed to assess the efficacy of concomitant cryoablation in patients scheduled for MV surgery. METHODS: Between 2012 and 2020, 242 adult patients who underwent MV surgery and concomitant cryoablation were included. Data on rhythm, medication status and clinical events were assessed at 3, 6 and 12 months, then annually thereafter. RESULTS: Early mortality was 0.4%. The mean follow-up period duration was 43.9 months. The survival rates at 1, 3 and 5 years were 97.3%, 94.3% and 87.7%, respectively. The rates of freedom from atrial arrhythmia paroxysms at 1, 3 and 5 years were 79.0%, 64.0% and 60.5%, respectively. Atrial arrhythmia recurrence was associated with isolated left atrial lesion set (P = 0.038), large right atrial size (P = 0.002), lower surgeon experience (P = 0.003) and atrial fibrillation paroxysms in the early postoperative period (P = 0.002). CONCLUSIONS: Concomitant cryoablation during MV surgery is a safe and reproducible technique. The procedure provides acceptable freedom from atrial arrhythmias recurrences during long-term follow-up. The biatrial lesion set has advantages over the left atrium pattern in terms of atrial arrhythmias freedom. Surgeon experience significantly influences atrial fibrillation ablation success. Randomized trials are needed to compare radiofrequency and cryoablation.

Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches.

In our previous study, we showed that discarded cardiac tissue from the right atrial appendage and right ventricular myocardium is an available source of functional endothelial and smooth muscle cells for regenerative medicine and tissue engineering. In the study, we aimed to find out what benefits are given by vascular cells from cardiac explants used for seeding on vascular patches engrafted to repair vascular defects in vivo. Additionally, to make the application of these cells safer in regenerative medicine we tested an in vitro approach that arrested mitotic division to avoid the potential tumorigenic effect of dividing cells. A tissue-engineered construction in the form of a patch based on a polycaprolactone-gelatin scaffold and seeded with endothelial and smooth muscle cells was implanted into the abdominal aorta of immunodeficient SCID mice. Aortic patency was assessed using ultrasound, MRI, immunohistochemical and histological staining. Endothelial and smooth muscle cells were treated with mitomycin C at a therapeutic concentration of 10 µg/ml for 2 h with subsequent analysis of cell proliferation and function. The absence of the tumorigenic effect of mitomycin C-treated cells, as well as their angiogenic potential, was examined by injecting them into immunodeficient mice. Cell-containing patches engrafted in the abdominal aorta of immunodeficient mice form the vessel wall loaded with the appropriate cells and extracellular matrix, and do not interfere with normal patency. Endothelial and smooth muscle cells treated with mitomycin C show no tumorigenic effect in the SCID immunodeficient mouse model. During in vitro experiments, we have shown that treatment with mitomycin C does not lead to a decrease in cell viability. Despite the absence of proliferation, mitomycin C-treated vascular cells retain specific cell markers, produce specific extracellular matrix, and demonstrate the ability to stimulate angiogenesis in vivo. We pioneered an approach to arresting cell division with mitomycin C in endothelial and smooth muscle cells from cardiac explant, which prevents the risk of malignancy from dividing cells in vascular surgery. We believe that this approach to the fabrication of tissue-engineered constructs based on mitotically inactivated cells from waste postoperative material may be valuable to bring closer the development of safe cell products for regenerative medicine.

Adipokine Levels in Men with Coronary Atherosclerosis on the Background of Abdominal Obesity.

Background. Obesity is associated with dyslipidemia, and excess body fat is associated with unfavorable levels of adipokines and markers of inflammation. The goal of research. To study the level of adipokines and markers of inflammation, their associations with unstable atherosclerotic plaques in men with coronary atherosclerosis on the background of abdominal obesity. Materials and methods. The study involved 82 men aged 40-77 years with coronary atherosclerosis after endarterectomy from the coronary arteries. We divided all men into two groups: 37 men (45.1%) with unstable atherosclerotic plaques, and 45 men (54.9%) who had stable plaques. Obesity was established at a BMI of ≥30 kg/m2. The levels of adipokines and markers of inflammation in the blood were determined by multiplex analysis. Results. In patients with obesity and unstable plaques, the levels of C-peptide, TNFa and IL-6 were 1.8, 1.6, and 2.8 times higher, respectively, than in patients with obesity and stable plaques. The chance of having an unstable plaque increases with an increase in TNFa by 49% in obese patients and decreases with an increase in insulin by 3% in non-obese patients. Conclusions. In men with coronary atherosclerosis and obesity, unstable atherosclerotic plaques in the coronary arteries are directly associated with the level of TNF-α.

Existing and Emerging Approaches to Risk Assessment in Patients with Ascending Thoracic Aortic Dilatation.

Ascending thoracic aortic aneurysm is a life-threatening disease, which is difficult to detect prior to the occurrence of a catastrophe. Epidemiology patterns of ascending thoracic aortic dilations/aneurysms remain understudied, whereas the risk assessment of it may be improved. The electronic databases PubMed/Medline 1966-2022, Web of Science 1975-2022, Scopus 1975-2022, and RSCI 1994-2022 were searched. The current guidelines recommend a purely aortic diameter-based assessment of the thoracic aortic aneurysm risk, but over 80% of the ascending aorta dissections occur at a size that is lower than the recommended threshold of 55 mm. Moreover, a 55 mm diameter criterion could exclude a vast majority (up to 99%) of the patients from preventive surgery. The authors review several visualization-based and alternative approaches which are proposed to better predict the risk of dissection in patients with borderline dilated thoracic aorta. The imaging-based assessments of the biomechanical aortic properties, the Young's elastic modulus, the Windkessel function, compliance, distensibility, wall shear stress, pulse wave velocity, and some other parameters have been proposed to improve the risk assessment in patients with ascending thoracic aortic aneurysm. While the authors do not argue for shifting the diameter threshold to the left, they emphasize the need for more personalized solutions that integrate the imaging data with the patient's genotypes and phenotypes in this heterogeneous pathology.

Antimitochondrial autoantibodies in pemphigus vulgaris: a missing link in disease pathophysiology.

A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3(-/-) mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3(-/-) KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.

Upregulation of nuclear factor-kappaB expression by SLURP-1 is mediated by alpha7-nicotinic acetylcholine receptor and involves both ionic events and activation of protein kinases.

SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a novel auto/paracrine cholinergic peptide that can bind to α(7)-nicotinic acetylcholine receptor (nAChR), a high Ca(2+)-permeable ion channel coupled to regulation of nuclear factor-κB (NF-κB) expression. Elucidation of intracellular signaling events elicited by SLURP-1 is crucial for understanding the molecular mechanism of functioning of this novel hormone-like peptide that alters vital cell functions and can protect from tumorigenic transformation. In this study, we sought to dissect out the role of α(7)-nAChR in mediating the biologic effects of recombinant SLURP-1 on the immortalized line of human oral keratinocytes Het-1A. A multifold upregulation of the NF-κB expression at the mRNA and protein levels by SLURP-1 was only slightly diminished due to elimination of Na(+), whereas in Ca(2+)-free medium the effect of SLURP-1 was inhibited by >50%. Both in the absence of extracellular Ca(2+) and in the presence of Cd(2+) or Zn(2+), the SLURP-1-dependent elevation of NF-κB was almost completely blocked by inhibiting MEK1 activity. Downstream of α(7)-nAChR, the SLURP-1 signaling coupled to upregulation of NF-κB also involved Jak2 as well as Ca(2+)/calmodulin-dependent kinase II (CaMKII) and protein kinase C (PKC), whose inhibition significantly (P < 0.05) reduced the SLURP-1-induced upregulation of NF-κB. The obtained results indicated that activation of α(7)-nAChR by SLURP-1 leads to upregulation of the NF-κB gene expression due to activation of the Raf-1/MEK1/ERK1/2 cascade that proceeds via two complementary signaling pathways. One is mediated by the Ca(2+)-entry dependent CaMKII/PKC activation and another one by Ca(2+)-independent involvement of Jak2. Thus, there exists a previously not appreciated network of noncanonical auto/paracrine ligands of nAChR of the Ly-6 protein family, which merits further investigations.

Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis.

Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a 'tombstone' appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering.

Receptor-mediated tobacco toxicity: acceleration of sequential expression of alpha5 and alpha7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke.

Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca(2+) influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca(2+) permeability, such as alpha5-containing alpha3 nAChR and alpha7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the alpha5 and alpha7 expression in KCs, which could be abolished by mecamylamine and alpha-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: alpha3(beta2/beta4) > alpha3(beta2/beta4)alpha5 > alpha7 > alpha7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the alpha3(beta2/beta4) to alpha3(beta2/beta4)alpha5 nAChR transition predominantly involved protein kinase C, alpha3(beta2/beta4)alpha5 to alpha7 nAChR transition-Ca(2+)/calmodulin-dependent protein kinase II and p38 MAPK, and alpha7 self-up-regulation-the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca(2+) permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products.

Central role of alpha7 nicotinic receptor in differentiation of the stratified squamous epithelium.

Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in nonneuronal locations, but their functions remain unknown. We found that keratinocyte alpha7 nAChR controls homeostasis and terminal differentiation of epidermal keratinocytes required for formation of the skin barrier. The effects of functional inactivation of alpha7 nAChR on keratinocyte cell cycle progression, differentiation, and apoptosis were studied in cell monolayers treated with alpha-bungarotoxin or antisense oligonucleotides and in the skin of Acra7 homozygous mice lacking alpha7 nAChR channels. Elimination of the alpha7 signaling pathway blocked nicotine-induced influx of 45Ca2+ and also inhibited terminal differentiation of these cells at the transcriptional and/or translational level. On the other hand, inhibition of the alpha7 nAChR pathway favored cell cycle progression. In the epidermis of alpha7-/- mice, the abnormalities in keratinocyte gene expression were associated with phenotypic changes characteristic of delayed epidermal turnover. The lack of alpha7 was associated with up-regulated expression of the alpha3 containing nAChR channels that lack alpha5 subunit, and both homomeric alpha9- and heteromeric alpha9alpha10-made nAChRs. Thus, this study demonstrates that ACh signaling through alpha7 nAChR channels controls late stages of keratinocyte development in the epidermis by regulating expression of the cell cycle progression, apoptosis, and terminal differentiation genes and that these effects are mediated, at least in part, by alterations in transmembrane Ca2+ influx.

Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of alpha7 nicotinic receptor in oral keratinocytes.

The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco-derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly alpha7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into alpha7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of alpha7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real-time polymerase chain reaction (PCR) and In-cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT-3. These effects could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the alpha7 antagonist alpha-bungarotoxin (alphaBTX) or transfected with anti-alpha7 small interfering RNA (siRNA-alpha7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ERK steps mediated alpha7-dependent up-regulation of STAT-3. Targeted mutation of the alpha7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT-3 caused by ADSS or pure nicotine was mediated by janus-activated kinase (JAK)-2. Activation of JAK-2/STAT-3 pathway could be prevented by alphaBTX or siRNA-alpha7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from alpha7, which proceeds via two complementary pathways. The Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT-3, whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Elucidation of this novel mechanism of nicotine-dependent nuclear transactivation of STAT-3 identifies oral alpha7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.

SLURP-1 and -2 in normal, immortalized and malignant oral keratinocytes.

The secreted mammalian Ly-6/urokinase plasminogen activator receptor-related proteins (SLURP)-1 and -2 are produced by keratinocytes comprising the mucocutaneous epithelium. They regulate in autocrine and paracrine ways cell growth and differentiation through the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. Keratinocyte nAChRs are targeted by tobacco-derived carcinogenic nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) that can induce tumorigenic transformation of Het-1A keratinocytes. In this study we asked if SLURPs could abolish tumorigenic effects of nitrosamines. Preincubation with either recombinant SLURP-1 or -2 in both cases considerably reduced the number of colonies in soft agar, and the number of tumor nodules >0.5 cm in diameter in Nu/Nu mice produced by Het-1A cells treated with nitrosamines. The levels of SLURP-1 and -2 mRNA transcripts in nitrosamine-transformed Het-1A cells as well as in the tumor cell lines SCC-25 and FaDu were significantly (p<0.05) less compared to normal gingival keratinocytes, which are probably the major source of the secreted SLURPs found in a sample of human saliva. The expression of SLURPs was decreased due to gene silencing of different nAChR alpha subunits with small hairpin RNA, suggesting that a positive feedback regulation is altered in malignant cells. Thus, SLURP-1 and -2 are efficient autocrine and paracrine ligands of keratinocyte nAChRs capable of preventing tobacco nitrosamine-induced malignant transformation of oral cells. These "proof-of-concept" preliminary results have salient clinical implications.

Receptor-mediated tobacco toxicity: alterations of the NF-kappaB expression and activity downstream of alpha7 nicotinic receptor in oral keratinocytes.

To gain a mechanistic insight into nicotinic receptor-dependent morbidity of tobacco products in the oral cavity, we studied effects of exposures of normal human oral keratinocytes (KCs) for 24 h to environmental tobacco smoke (ETS) vs. equivalent concentration of pure nicotine. The exposed KCs showed a multifold increase of nuclear factor-kappaB (NF-kappaB) at the mRNA and protein levels, which could be significantly (p<0.05) diminished by alpha-bungarotoxin or transfection with anti-alpha7 small interfering RNA. An increased protein-binding activity of NF-kappaB also could be prevented by blocking alpha7 signaling. The use of pathway inhibitors demonstrated that the Ras/Raf-1/MEK1/ERK steps mediated alpha7-dependent upregulation of NF-kappaB. Thus, exposure of KCs to tobacco may lead to the pathobiologic effects via an intracellular signaling pathway downstream of alpha7 that proceeds through the Ras/Raf-1/MEK1/ERK steps leading to upregulated expression and transactivation of NF-kappaB.