Interactions between patterns of multimorbidity and functional status among hospitalized older patients: a novel approach using cluster analysis and association rule mining.

BACKGROUND: Multimorbidity (MM) is generally defined as the presence of 2 or more chronic diseases in the same patient and seems to be frequently associated with frailty and poor quality of life. However, the complex interplay between MM and functional status in hospitalized older patients has not been fully elucidated so far. Here, we implemented a 2-step approach, combining cluster analysis and association rule mining to explore how patterns of MM and disease associations change as a function of disability. METHODS: This retrospective cohort study included 3366 hospitalized older patients discharged from acute care units of Ancona and Cosenza sites of Italian National Institute on Aging (INRCA-IRCCS) between 2011 and 2017. Cluster analysis and association rule mining (ARM) were used to explore patterns of MM and disease associations in the whole population and after stratifying by dependency in activities of daily living (ADL) at discharge. Sensitivity analyses in men and women were conducted to test for robustness of study findings. RESULTS: Out of 3366 included patients, 78% were multimorbid. According to functional status, 22.2% of patients had no disability in ADL (functionally independent group), 22.7% had 1 ADL dependency (mildly dependent group), and 57.4% 2 or more ADL impaired (moderately-severely dependent group). Two main MM clusters were identified in the whole general population and in single ADL groups. ARM revealed interesting within-cluster disease associations, characterized by high lift and confidence. Specifically, in the functionally independent group, the most significant ones involved atrial fibrillation (AF)-anemia and chronic kidney disease (CKD) (lift = 2.32), followed by coronary artery disease (CAD)-AF and heart failure (HF) (lift = 2.29); in patients with moderate-severe ADL disability, the most significant ARM involved CAD-HF and AF (lift = 1.97), thyroid dysfunction and AF (lift = 1.75), cerebrovascular disease (CVD)-CAD and AF (lift = 1.55), and hypertension-anemia and CKD (lift = 1.43). CONCLUSIONS: Hospitalized older patients have high rates of MM and functional impairment. Combining cluster analysis to ARM may assist physicians in discovering unexpected disease associations in patients with different ADL status. This could be relevant in the view of individuating personalized diagnostic and therapeutic approaches, according to the modern principles of precision medicine.

Association of Torquetenovirus Viremia with Physical Frailty and Cognitive Impairment in Three Independent European Cohorts.

INTRODUCTION: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. METHODS: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). RESULTS: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. CONCLUSIONS: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals.

Blood circulating bacterial DNA in hospitalized old COVID-19 patients.

BACKGROUND: Coronavirus disease COVID-19 is a heterogeneous condition caused by SARS-CoV-2 infection. Generally, it is characterized by interstitial pneumonia that can lead to impaired gas-exchange, acute respiratory failure, and death, although a complex disorder of multi-organ dysfunction has also been described. The pathogenesis is complex, and a variable combination of factors has been described in critically ill patients. COVID-19 is a particular risk for older persons, particularly those with frailty and comorbidities. Blood bacterial DNA has been reported in both physiological and pathological conditions and has been associated with some haematological and laboratory parameters but, to date, no study has characterized it in hospitalized old COVID-19 patients The present study aimed to establish an association between blood bacterial DNA (BB-DNA) and clinical severity in old COVID-19 patients. RESULTS: BB-DNA levels were determined, by quantitative real-time PCRs targeting the 16S rRNA gene, in 149 hospitalized older patients (age range 65-99 years) with COVID-19. Clinical data, including symptoms and signs of infection, frailty status, and comorbidities, were assessed. BB-DNA was increased in deceased patients compared to discharged ones, and Cox regression analysis confirmed an association between BB-DNA and in-hospital mortality. Furthermore, BB-DNA was positively associated with the neutrophil count and negatively associated with plasma IFN-alpha. Additionally, BB-DNA was associated with diabetes. CONCLUSIONS: The association of BB-DNA with mortality, immune-inflammatory parameters and diabetes in hospitalized COVID-19 patients suggests its potential role as a biomarker of unfavourable outcomes of the disease, thus it could be proposed as a novel prognostic marker in the assessment of acute COVID-19 disease.

Effects of Human LAV-BPIFB4 Gene Therapy on the Epigenetic Clock and Health of Aged Mice.

The homozygous genotype of the Longevity-Associated Variant (LAV) in Bactericidal/Permeability-Increasing Fold-Containing Family B member 4 (BPIFB4) is enriched in long-living individuals of three independent populations and its genetic transfer in C57BL/6J mice showed a delay in frailty progression and improvement of several biomarkers of aging and multiple aspects of health. The C57BL/6J strain is a suitable model for studying therapies aimed at extending healthy aging and longevity due to its relatively short lifespan and the availability of aging biomarkers. Epigenetic clocks based on DNA methylation profiles are reliable molecular biomarkers of aging, while frailty measurement tools are used to evaluate overall health during aging. In this study, we show that the systemic gene transfer of LAV-BPIFB4 in aged C57BL/6J mice was associated with a significant reduction in the epigenetic clock-based biological age, as measured by a three CpG clock method. Furthermore, LAV-BPIFB4 gene transfer resulted in an improvement of the Vitality Score with a reduction in the Frailty Index. These findings further support the use of LAV-BPIFB4 gene therapy to induce beneficial effects on epigenetic mechanisms associated with aging and frailty in aged mice, with potential implications for future therapies to prevent frailty in humans.

Effect of Cytomegalovirus Reactivation on Inflammatory Status and Mortality of Older COVID-19 Patients.

Herpesviridae reactivation such as cytomegalovirus (CMV) has been described in severe COVID-19 (COronaVIrusDisease-2019). This study aimed to understand if CMV reactivation in older COVID-19 patients is associated with increased inflammation and in-hospital mortality. In an observational single-center cohort study, 156 geriatric COVID-19 patients were screened for CMV reactivation by RT-PCR. Participants underwent a comprehensive clinical investigation that included medical history, functional evaluation, laboratory tests and cytokine assays (TNF-α, IFN-α, IL-6, IL-10) at hospital admission. In 19 (12.2%) of 156 COVID-19 patients, CMV reactivation was detected. Multivariate Cox regression models showed that in-hospital mortality significantly increased among CMV positive patients younger than 87 years (HR: 9.94, 95% CI: 1.66-59.50). Other factors associated with in-hospital mortality were C-reactive protein (HR: 1.17, 95% CI: 1.05-1.30), neutrophil count (HR: 1.20, 95% CI: 1.01-1.42) and clinical frailty scale (HR:1.54, 95% CI: 1.04-2.28). In patients older than 87 years, neutrophil count (HR: 1.13, 95% CI: 1.05-1.21) and age (HR: 1.15, 95% CI: 1.01-1.31) were independently associated with in-hospital mortality. CMV reactivation was also correlated with increased IFN-α and TNF-α serum levels, but not with IL-6 and IL-10 serum changes. In conclusion, CMV reactivation was an independent risk factor for in-hospital mortality in COVID-19 patients younger than 87 years old, but not in nonagenarians.

Bacterial DNAemia in Alzheimer's Disease and Mild Cognitive Impairment: Association with Cognitive Decline, Plasma BDNF Levels, and Inflammatory Response.

Microbial dysbiosis (MD) provokes gut barrier alterations and bacterial translocation in the bloodstream. The increased blood bacterial DNA (BB-DNA) may promote peripheral- and neuro-inflammation, contributing to cognitive impairment. MD also influences brain-derived neurotrophic factor (BDNF) production, whose alterations contribute to the etiopathogenesis of Alzheimer's disease (AD). The purpose of this study is to measure BB-DNA in healthy elderly controls (EC), and in patients with mild cognitive impairment (MCI) and AD to explore the effect on plasma BDNF levels (pBDNF), the inflammatory response, and the association with cognitive decline during a two-year follow-up. Baseline BB-DNA and pBDNF were significantly higher in MCI and AD than in EC. BB-DNA was positively correlated with pBDNF in AD, plasma Tumor necrosis factor-alpha (TNF-α), and Interleukin-10 (IL-10) levels in MCI. AD patients with BB-DNA values above the 50th percentile had lower baseline Mini-Mental State Examination (MMSE). After a two-year follow-up, AD patients with the highest BB-DNA tertile had a worse cognitive decline, while higher BB-DNA levels were associated with higher TNF-α and lower IL-10 in MCI. Our study demonstrates that, in early AD, the higher the BB-DNA levels, the higher the pBDNF levels, suggesting a defensive attempt; BB-DNA seems to play a role in the AD severity/progression; in MCI, higher BB-DNA may trigger an increased inflammatory response.

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer.

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated p16 expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.

New Evidence and Insights on Dalbavancin and Wound Healing in a Mouse Model of Skin Infection.

Dalbavancin is an effective antibiotic that is widely used to treat skin infection. Our aim was to determine the effect of dalbavancin administration on wound healing compared to that of vancomycin and to elucidate if epidermal growth factor receptor (EGFR), matrix metalloproteinase 1 (MMP-1), MMP-9, and vascular endothelial growth factor (VEGF) could be involved in its therapeutic mechanism. A mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection was established. Mice were treated daily with vancomycin (10 mg/kg) and weekly with dalbavancin at day 1 (20 mg/kg) and day 8 (10 mg/kg). After 14 days, wounds were excised, and bacterial counts were performed. Wound healing was assessed by histological and immunohistochemical staining, followed by protein extraction and immunoblotting. Our microbiological results confirmed that both dalbavancin and vancomycin are effective in reducing the bacterial load in wounds. The dalbavancin group showed a strong effect compared with infected untreated animals and the vancomycin-treated group. The wounds treated with dalbavancin showed robust epidermal coverage with reconstitution of the regular and keratinized epidermal lining and well-organized granulation tissue with numerous blood vessels, although slightly less than that in the uninfected group. While in the vancomycin-treated group the epithelium appeared, in general, still hypertrophic, the granulation tissue appeared even less organized. We observed elevated EGFR and VEGF expression in both treated groups, although it was higher in dalbavancin-treated mice. MMP-1 and MMP-9 were decreased in uninfected tissue and in both treated tissues compared with untreated infected wounds. This study showed faster healing with dalbavancin treatment that might be associated with higher EGFR and VEGF levels.

Aging-Related Expression of Twinfilin-1 Regulates Cholangiocyte Biological Response to Injury.

Disorders of the biliary tree develop and progress differently according to patient age. It is currently not known whether the aging process affects the response to injury of cholangiocytes. The aim of this study was to identify molecular pathways associated with cholangiocyte aging and to determine their effects in the biological response to injury of biliary cells. A panel of microRNAs (miRs) involved in aging processes was evaluated in cholangiocytes of young and old mice (2 months and 22 months of age, respectively) and subjected to a model of sclerosing cholangitis. Intracellular pathways that are common to elevated miRs were identified by in silico analysis. Cell proliferation and senescence were evaluated in Twinfilin-1 (Twf1) knocked-down cells. In vivo, senescence-accelerated prone mice (Samp8, a model for accelerated aging), Twf1-/- , or their respective controls were subjected to DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine). Cholangiocytes from DDC-treated mice showed up-regulation of a panel of aging-related miRs. Twf1 was identified by in silico analysis as a common target of the up-regulated miRs. Twf1 expression was increased both in aged and diseased cholangiocytes, and in human cholangiopathies. Knock-down of Twf1 in cholangiocytes reduced cell proliferation. Senescence and senescence-associated secretory phenotype marker expression increased in Twf1 knocked-down cholangiocytes following pro-proliferative and pro-senescent (10-day lipopolysaccharide) stimulation. In vivo, Samp8 mice showed increased biliary proliferation, fibrosis, and Twf1 protein expression level, whereas Twf1-/- had a tendency toward lower biliary proliferation and fibrosis following DDC administration compared with control animals. Conclusion: We identified Twf1 as an important mediator of both cholangiocyte adaptation to aging processes and response to injury. Our data suggest that disease and aging might share common intracellular pathways.

Is cellular senescence involved in cystic fibrosis?

Pulmonary disease is the main cause of the morbidity and mortality of patients affected by cystic fibrosis (CF). The lung pathology is dominated by excessive recruitment of neutrophils followed by an exaggerated inflammatory process that has also been reported to occur in the absence of apparent pathogenic infections. Airway surface dehydration and mucus accumulation are the driving forces of this process. The continuous release of reactive oxygen species and proteases by neutrophils contributes to tissue damage, which eventually leads to respiratory insufficiency. CF has been considered a paediatric problem for several decades. Nevertheless, during the last 40 years, therapeutic options for CF have been greatly improved, turning CF into a chronic disease and extending the life expectancy of patients. Unfortunately, chronic inflammatory processes, which are characterized by a substantial release of cytokines and chemokines, along with ROS and proteases, can accelerate cellular senescence, leading to further complications in adulthood. The alterations and mechanisms downstream of CFTR functional defects that can stimulate cellular senescence remain unclear. However, while there are correlative data suggesting that cellular senescence may be implicated in CF, a causal or consequential relationship between cellular senescence and CF is still far from being established. Senescence can be both beneficial and detrimental. Senescence may suppress bacterial infections and cooperate with tissue repair. Additionally, it may act as an effective anticancer mechanism. However, it may also promote a pro-inflammatory environment, thereby damaging tissues and leading to chronic age-related diseases. In this review, we present the most current knowledge on cellular senescence and contextualize its possible involvement in CF.

FKBP5 rs4713916: A Potential Genetic Predictor of Interindividual Different Response to Inhaled Corticosteroids in Patients with Chronic Obstructive Pulmonary Disease in a Real-Life Setting.

Background: Chronic obstructive pulmonary disease (COPD) is a common, preventable, and manageable lung disease characterized by large heterogeneity in disease presentation and grades impairment. Inhaled corticosteroids (ICS) are commonly used to manage COPD/COPD-exacerbation. The patient's response is characterized by interindividual variability without disease progression/survival modification. Objectives: We hypothesize that a therapeutic intervention may be more effective if single nucleotide polymorphisms (SNPs) are investigated. Methods: In 71 COPD patients under pulmonary rehabilitation, a small number of powerful SNPs, selected according to current literature, were analyzed; namely the glucocorticoid receptor gene NR3C1 (rs6190/rs6189/rs41423247), the glucocorticoid-induced transcript 1 gene (GLCCI1 rs37972), and the related co-chaperone FKBP5 gene (rs4713916). MDR1 rs2032582 was also evaluated. Lung function outcomes were assessed. Results: A significant association with functional outcomes, namely FEV1 (forced expiration volume/one second) and 6MWD (six-minutes walking distance), was found for rs4713916 and weakly for rs37972. The genotype rs4713916(GA) and, in a lesser extent, the genotype rs37972(TT), were more favorable than the wild-type. Conclusions: Our study supports a possible picture of pharmacogenomic control for COPD intervention. rs4713916 and, possibly, rs37972 may be useful predictors of clinical outcome. These results may help to tailor an optimal dose for individual COPD patients based on their genetic makeup.

Anti-inflammatory Activity of Tocotrienols in Age-related Pathologies: A SASPected Involvement of Cellular Senescence.

Tocotrienols (T3) have been shown to represent a very important part of the vitamin E family since they have opened new opportunities to prevent or treat a multitude of age-related chronic diseases. The beneficial effects of T3 include the amelioration of lipid profile, the promotion of Nrf2 mediated cytoprotective activity and the suppression of inflammation. All these effects may be the consequence of the ability of T3 to target multiple pathways. We here propose that these effects may be the result of a single target of T3, namely senescent cells. Indeed, T3 may act by a direct suppression of the senescence-associated secretory phenotype (SASP) produced by senescent cells, mediated by inhibition of NF-kB and mTOR, or may potentially remove the origin of the SASP trough senolysis (selective death of senescent cells). Further studies addressed to investigate the impact of T3 on cellular senescence "in vitro" as well as in experimental models of age-related diseases "in vivo" are clearly encouraged.

Inducers of Senescence, Toxic Compounds, and Senolytics: The Multiple Faces of Nrf2-Activating Phytochemicals in Cancer Adjuvant Therapy.

The reactivation of senescence in cancer and the subsequent clearance of senescent cells are suggested as therapeutic intervention in the eradication of cancer. Several natural compounds that activate Nrf2 (nuclear factor erythroid-derived 2-related factor 2) pathway, which is involved in complex cytoprotective responses, have been paradoxically shown to induce cell death or senescence in cancer. Promoting the cytoprotective Nrf2 pathway may be desirable for chemoprevention, but it might be detrimental in later stages and advanced cancers. However, senolytic activity shown by some Nrf2-activating compounds could be used to target senescent cancer cells (particularly in aged immune-depressed organisms) that escape immunosurveillance. We herein describe in vitro and in vivo effects of fifteen Nrf2-interacting natural compounds (tocotrienols, curcumin, epigallocatechin gallate, quercetin, genistein, resveratrol, silybin, phenethyl isothiocyanate, sulforaphane, triptolide, allicin, berberine, piperlongumine, fisetin, and phloretin) on cellular senescence and discuss their use in adjuvant cancer therapy. In light of available literature, it can be concluded that the meaning and the potential of adjuvant therapy with natural compounds in humans remain unclear, also taking into account the existence of few clinical trials mostly characterized by uncertain results. Further studies are needed to investigate the therapeutic potential of those compounds that display senolytic activity.

The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model.

The amnion muscle combined graft (AMCG) conduits showed good clinical results in peripheral nerves gap repair. It combines the human amniotic membrane with autologous skeletal muscle fibres. These results seem attributable to the biological characteristics of human amniotic membrane: Pluripotency, anti-inflammatory and low immunogenicity.We here evaluate the final outcome of nerve regeneration morphologically and functionally, across the AMCG compared to nerve autograft. Fourteen Wistar rats were divided into two groups: In Group A, including 6 rats, the left forelimb was treated performing a 1.5 cm length gap on median nerve that was then reconstructed with a reverse autograft. In Group B, including 8 rats, the gap was reconstructed with AMCG. Functional results were evaluated at 30, 60 and 90 days performing grasping tests. Morphological and stereological analyses were performed at T90 using high-resolution light microscopy and design-based stereology. The AMCG conduits revealed nerve fibres regeneration and functional recovery. Functional recovery was observed in both groups with AMCG conduits group showing lower values and a regeneration of median nerves with more myelinated fibres with the same axon size, but thinner myelin than the autograft group. Though the autograft remains the gold standard to restore wide nerve gaps, the AMCG conduit has proved to be effective in enabling nerve regeneration through a critical rat's nerve gap of 15 mm. These findings empirically support the great clinical results obtained using AMCG conduit to restore traumatic nerve's gap from 3 to 6 cm of mixed forearm nerves.

Allyl Isothiocyanate Exhibits No Anticancer Activity in MDA-MB-231 Breast Cancer Cells.

It was reported recently that allyl isothiocyanate (AITC) could inhibit various types of cancer cell growth. In the present study, we further investigated whether AITC could inhibit the growth of human breast cancer cells. Unexpectedly, we found that AITC did not inhibit, rather slightly promoted, the proliferation of MDA-MB-231 breast cancer cells, although it did have inhibitory effect on MCF-7 breast cancer cells. Cytofluorimetric analysis revealed that AITC (10 µM) did not induce apoptosis and cell cycle arrest in MDA-MB-231 cells. In addition, AITC significantly (p < 0.05) increased the expression of BCL-2 and mTOR genes and Beclin-1 protein in MDA-MB-231 cells. No significant changes in expression of PRKAA1 and PER2 genes, Caspase-8, Caspase-9, PARP, p-mTOR, and NF-κB p65 proteins were observed in these AITC-treated cells. Importantly, AITC displayed cytotoxic effect on MCF-10A human breast epithelial cell line. These observations suggest that AITC may not have inhibitory activity in MDA-MB-231 breast cancer cells. This in vitro study warrants more preclinical and clinical studies on the beneficial and harmful effects of AITC in healthy and cancer cells.

Role of Daptomycin on Burn Wound Healing in an Animal Methicillin-Resistant Staphylococcus aureus Infection Model.

Prolonged hospitalization and antibiotic therapy are risk factors for the development of methicillin-resistant Staphylococcus aureus (MRSA) infections in thermal burn patients. We used a rat model to study the in vivo efficacy of daptomycin in the treatment of burn wound infections by S. aureus, and we evaluated the wound healing process through morphological and immunohistochemical analysis. A copper bar heated in boiling water was applied on a paraspinal site of each rat, resulting in two full-thickness burns. A small gauze was placed over each burn and inoculated with 5 × 107 CFU of S. aureus ATCC 43300. The study included two uninfected control groups with and without daptomycin treatment, an infected control group that did not receive any treatment, and two infected groups treated, respectively, with intraperitoneal daptomycin and teicoplanin. The main outcome measures were quantitative culture, histological evaluation of tissue repair, and immunohistochemical expression of wound healing markers: epidermal growth factor receptor (EGFR) and fibroblast growth factor 2 (FGF-2). The highest inhibition of infection was achieved in the group that received daptomycin, which reduced the bacterial load from 107 CFU/ml to about 103 CFU/g (P < 0.01). The groups treated with daptomycin showed better overall healing with epithelialization and significantly higher collagen scores than the other groups, and these findings were also confirmed by immunohistochemical data. In conclusion, our results support the hypothesis that daptomycin is an important modulator of wound repair by possibly reducing hypertrophic burn scar formation.

Different transcriptional profiling between senescent and non-senescent human coronary artery endothelial cells (HCAECs) by Omeprazole and Lansoprazole treatment.

Recent evidence suggests that high dose and/or long term use of proton pump inhibitors (PPIs) may increase the risk of adverse cardiovascular events in older patients, but mechanisms underlying these detrimental effects are not known. Taking into account that the senescent endothelial cells have been implicated in the genesis or promotion of age-related cardiovascular disease, we hypothesized an active role of PPIs in senescent cells. The aim of this study is to investigate the changes in gene expression occurring in senescent and non-senescent human coronary artery endothelial cells (HCAECs) following Omeprazole (OPZ) or Lansoprazole (LPZ) treatment. Here, we show that atherogenic response is among the most regulated processes in PPI-treated HCAECs. PPIs induced down-regulation of anti-atherogenic chemokines (CXCL11, CXCL12 and CX3CL1) in senescent but not in non-senescent cells, while the same chemokines were up-regulated in untreated senescent cells. These findings support the hypothesis that up-regulated anti-atherogenic chemokines may represent a defensive mechanism against atherosclerosis during cellular senescence, and suggest that PPIs could activate pro-atherogenic pathways by changing the secretory phenotype of senescent HCAECs. Moreover, the genes coding for fatty acid binding protein 4 (FABP4) and piezo-type mechanosensitive ion channel component 2 (PIEZO2) were modulated by PPIs treatment with respect to untreated cells. In conclusions, our results show that long-term and high dose use of PPI could change the secretory phenotype of senescent cells, suggesting one of the potential mechanisms by which use of PPI can increase adverse outcomes in older subjects.

Gene Expression, Oxidative Stress, and Senescence of Primary Coronary Endothelial Cells Exposed to Postprandial Serum of Healthy Adult and Elderly Volunteers after Oven-Cooked Meat Meals.

Epidemiological studies have linked high consumption of meat with major age-related diseases including cardiovascular diseases. Abnormal postprandial increases in plasma lipids after a meat meal have been hypothesized among the pathogenetic mechanisms. However, it is still unknown if the postprandial serum derived after a normal meat meal is able to affect endothelial function, and if the type of meat and the age of the donors are critical factors. Here, we show the effects of postprandial sera derived from healthy adults and elderly volunteers who consumed meat meals on human coronary artery endothelial cell (HCAEC) oxidative stress, gene expression, DNA damage, and cellular senescence. We observed that a single exposure to postprandial serum induces a slight increase in ROS that is associated with modulation of gene expression pathways related to oxidative stress response and metabolism. The postprandial-induced increase in ROS is not associated with a measurable DNA oxidative damage. However, repeated exposure to postprandial serum induces an acceleration of cellular senescence. Taking into account the deleterious role of cellular senescence in age-related vascular diseases, the results suggest a new mechanism by which excessive meat consumption and time spent in postprandial state may affect health status during aging.

Increase of n-NOS and i-NOS in Rat Colon After Sacral Neuromodulation.

OBJECTIVE: Sacral neuromodulation (SNM) is proposed to treat different anorectal dysfunctions but its mechanism of action is not yet known. Our previous study demonstrated how SNM can significantly increase neuronal nitric oxide synthase NOS (n-NOS) and inducible NOS (i-NOS) expression in the anus and rectum of rats. There are no reports regarding the relation between SNM and NOS in colonic cells: our aim was to assess NOS expression in colonic rat model after SNM. MATERIALS AND METHODS: Twenty-six female Sprangue-Dawley rats were considered: group I, normal control rats; group II, sham treatment rats, in whom electrodes for electrical stimulation were placed in S1 foramen bilaterally and left in place, without performing neuromodulation; group III, rats in whom SNM was performed. After 14 days, the rats were sacrificed and we evaluated n-NOS and i-NOS in colonic specimens by immunohistochemistry and Western Blot analysis. RESULTS: Western Blot analysis showed that levels of n-NOS and i-NOS were higher in colon of the III group rats respect to the others; in particular, immunohistochemistry revealed that, after neuromodulation, n-NOS expression in the muscle cells and i-NOS expression in glandular epithelium and nervous cells were highly represented (p < 0.05). CONCLUSION: Our study showed that in colon, SNM is able to influence NO synthesis, activating n-NOS expression in muscle cells and i-NOS expression in glandular epithelium and nervous cells. Our study showed a complex colonic response to SNM. This experimental model could be applied to better understand the mechanism of action of SNM in bowel dysfunction.

Antiangiogenic and antitumor activities of berberine derivative NAX014 compound in a transgenic murine model of HER2/neu-positive mammary carcinoma.

Berberine (BBR) is a natural isoquinoline alkaloid with proven antiangiogenic and anticancer activities. We recently demonstrated that BBR and its synthetic derivative 13-(4-chlorophenylethyl)berberine iodide, NAX014, exert antiproliferative activity against HER2-overexpressing breast cancer cells, inducing apoptosis, modulating the expression of cell cycle checkpoint molecules involved in cell senescence, and reducing both HER2 expression and phosphorylation on tumor cells. In this study, we examined the anticancer properties of BBR and NAX014 in a transgenic mouse model which spontaneously develops HER2-positive mammary tumors. Repeated intraperitoneal injections of a safety dose (2.5mg/kg) of NAX014 delayed the development of tumors, reducing both the number and size of tumor masses. In vivo sidestream dark field videomicroscopy revealed a significant lower vessel density in mammary tumors from NAX014-treated mice in comparison with the control group. Immunohistochemical evaluation using CD34 antibody confirmed the reduced vessel density in NAX014 group. Statistically significant increase of senescence associated ß-galactosidase and p16 expression, and reduced expression of heparanase were observed in tumors from NAX014-treated mice than in tumors from control animals. Finally, NAX014 treatment decreased the level of perforine and granzyme mRNA in mammary tumors. Berberine did not show any statistically significant modulation in comparison with control mice. The results of the present study indicate that NAX014 is more effective than BBR in exerting anticancer activity delaying the development of mammary tumors in mice transgenic for the HER-2/neu oncogene. The antitumor efficacy of NAX014 is mainly related to its effect on tumor vascular network and on induction of tumor cell senescence.