Gaetana Costanza

Sortilin expression is essential for pro-nerve growth factor-induced apoptosis of rat vascular smooth muscle cells.

Background Sortilin, a member of the Vps10p-domain receptor family, has been demonstrated a key regulator in mediating cellular response to pro-neurotrophins. In the present study, we investigated the role of sortilin in the apoptotic pathway of vascular smooth muscle cells. Methods and Principal Findings Immunohistochemistry revealed that sortilin was barely detectable in human and rat normal young vessels, while its expression was increased in human fibroatheromatous plaques. Sortilin immunodetection was also marked in the neointima of the rat aorta fifteen days after ballooning. In vitro, rat aortic intimal cells expressed higher sortilin levels than normal media SMCs; sortilin was distributed in the cytoplasm and in correspondence of the cell membrane. After 48 h, pro-nerve growth factor (proNGF) induced the strong dose-dependent increase of intimal cell apoptosis and the accumulation of sortilin protein. ProNGF was a more potent apoptotic inducer than equimolar or even higher concentration of NGF, whereas brain derived neutrotrophic factor was ineffective. Targeted interfering RNA-mediated sortilin reduction counteracted proNGF-induced apoptosis without affecting p75NTR expression. ProNGF-induced apoptosis was associated to NF-κB down-regulation and bax increase. Inhibition of NF-κB activity increased intimal cell apoptosis that did not further increase with the addition of proNGF. Conclusions Our results indicate that sortilin expression characterizes human atheromatous lesions and rat aortic post-injury neointima, and suggest that sortilin represents an important regulator of proNGF-induced SMC apoptosis and arterial remodeling.

Propionyl-L-Carnitine is Efficacious in Ulcerative Colitis Through its Action on the Immune Function and Microvasculature.

Objectives:Microvascular endothelial dysfunction characterizes ulcerative colitis (UC), the most widespread form of inflammatory bowel disease. Intestinal mucosal microvessels in UC display aberrant expression of cell adhesion molecules (CAMs) and increased inflammatory cell recruitment. Propionyl-L-carnitine (PLC), an ester of L-carnitine required for the mitochondrial transport of fatty acids, ameliorates propionyl-CoA bioavailability and reduces oxidative stress in ischemic tissues. The present study aimed to document the efficacy of anti-oxidative stress properties of PLC in counteracting intestinal microvascular endothelial dysfunction and inflammation.Methods:To evaluate the efficacy in vivo, we analyzed the effects in intestinal biopsies of patients with mild-to-moderate UC receiving oral PLC co-treatment and in rat TNBS-induced colitis; in addition, we investigated antioxidant PLC action in TNF-α-stimulated human intestinal microvascular endothelial cells (HIMECs) in vitro.Results:Four-week PLC co-treatment reduced intestinal mucosal polymorph infiltration and CD4+ lymphocytes, ICAM-1+ and iNOS+ microvessels compared with placebo-treated patients with UC. Oral and intrarectal administration of PLC but not L-carnitine or propionate reduced intestinal damage and microvascular dysfunction in rat TNBS-induced acute and reactivated colitis. In cultured TNF-α-stimulated HIMECs, PLC restored β-oxidation and counteracted NADPH oxidase 4-generated oxidative stress-induced CAM expression and leukocyte adhesion. Inhibition of β-oxidation by L-aminocarnitine increased reactive oxygen species production and PLC beneficial effects on endothelial dysfunction and leukocyte adhesion. Finally, PLC reduced iNOS activity and nitric oxide accumulation in rat TNBS-induced colitis and in HIMEC cultures.Conclusions:Our results show that the beneficial antioxidant effect of PLC targeting intestinal microvasculature restores endothelial β-oxidation and function, and reduces mucosal inflammation in UC patients.

The relevance of piroxicam for the prevention and treatment of nonmelanoma skin cancer and its precursors

Piroxicam (PXM), a nonsteroidal anti-inflammatory drug, is an enolic benzothiazine and a potent member of the oxicam series. The drug suppresses the synthesis of proinflammatory enzymes, such as cyclo-oxygenases-1 and -2 (COX-1 and 2), downregulates the production of prostaglandins (PGs) and tromboxanes, and inhibits polyamines production by blocking ornithine decarboxylase induction involved in nonmelanoma skin carcinogenesis. In addition, PXM is able to induce tumor cell apoptosis and suppresses metalloproteinase 2 activities. Skin carcinogenesis is a multistep process in which the accumulation of genetic events leads to a gradually dysplastic cellular expression, deregulation of cell growth, and carcinomatous progression. COX-1 upregulation plays a significant role in PG and vascular epidermal growth factor production supporting tumor growth. Increased level of PGs in premalignant and/or malignant cutaneous tumors is also favored by upregulation of COX-2 and downregulation of the tumor suppressor gene 15-hydroxy-prostaglandin dehydrogenase. Chemoprevention can be a hopeful approach to inhibit carcinoma occurrence before an invasive tumor develops. The chemopreventive effect of nonsteroidal anti-inflammatory drugs on nonmelanoma skin cancers has been established. In this study, we highlighted the different modalities of action of PXM on the pathogenesis of nonmelanoma skin cancer, analyzing and evaluating binding modes and energies between COX-1 or COX-2 and PXM by protein–ligand molecular docking. Our clinical experience about the local use of PXM on actinic keratoses and field cancerization is also reported, confirming its efficacy as target therapy.

Antioxidant Treatment Prevents Serum Deprivation- and TNF-α-Induced Endothelial Dysfunction through the Inhibition of NADPH Oxidase 4 and the Restoration of β-Oxidation

Aims: Oxidative stress plays a pivotal role in the impaired endothelial function occurring in vascular diseases. Antioxidant strategies induce a clinical advantage in patients with endothelial dysfunction and atherosclerosis and protect from oxidative damage, but the underlying molecular mechanisms have been poorly evaluated. The aim of this study was to analyze the effects and mechanisms of action of antioxidant regimens on endothelial function. Methods and Results: Antioxidant efficacy of N-acetylcysteine, ascorbic acid and propionyl-L-carnitine was evaluated in serum-deprived and TNF-α-stimulated human umbilical vein endothelial cells in vitro. Cell adhesion molecule (CAM) expression was evaluated by blot and real-time PCR, and inflammatory cytokine secretion was evaluated by ELISA; leukocyte adhesion and reactive oxygen species assays and NADPH oxidase 4 isoform (Nox4) expression analyses by blots were also performed. Antioxidant pretreatment restored serum-deprived and TNF-α-induced impaired mitochondrial β-oxidation by reducing flavin adenine dinucleotide level and counteracting increased CAM and Nox4 expression, leukocyte adhesion and inflammatory cytokine secretion. Specific inhibition by plumbagin and siNox4 prevented TNF-α- and serum deprivation-induced detrimental effects, confirming that endothelial oxidative stress and inflammation were Nox4 dependent. Conclusions: Our findings documented Nox4 as a main actor in oxidative stress-induced endothelial dysfunction and further clarify the molecular basis of antioxidant treatment efficacy.

Tazarotene as alternative topical treatment for onychomycosis

Background Distal and lateral onychomycoses are the most frequent forms of onychomycosis, causing subungual hyperkeratosis that usually limits local penetration of antimycotic drugs. Tazarotene exerts anti-inflammatory and immune-modulating activities toward both infective agents and damaged keratinocytes. Given the well-documented efficacy of tazarotene on hyperkeratotic nail psoriasis, we investigated its therapeutic use in onychomycosis. Patients and methods We designed a preliminary open clinical trial in patients affected by distal and lateral subungual onychomycosis of the toenails and verified the fungistatic activity of tazarotene in vitro. Fifteen patients were treated with topical tazarotene 0.1% gel once per day for 12 weeks. Mycological cultures and potassium hydroxide stains of nail samples were performed at the beginning and at the end of the study. Treatment was considered effective when clinical healing and negative mycological culture were obtained. Onycholysis, nail bed discoloration, and subungual hyperkeratosis were measured using standardized methodologies and analyzed by means of Mann–Whitney test and analysis of variance. Fungistatic activity of tazarotene was evaluated by disk diffusion assay. Results Six patients (40%) reached a mycological cure on target nail samples already after 4 weeks of treatment. Complete clinical healing and negative cultures were reached in all patients at week 12, with a significant improvement of all clinical parameters of the infected nails. Disk diffusion assay after 48 hours of incubation with tazarotene solution showed a central area of inhibition in all examined fungal cultures. Conclusion Our results documented a good clinical outcome using topical tazarotene 0.1% gel in distal and lateral subungual onychomycosis and its fungistatic activity of tazarotene in vitro. The majority of patients appeared cured at a 6-month follow-up. The efficacy and safety of tazarotene must be confirmed on a larger number of patients, although already documented in nail psoriasis patients often affected by onychomycosis.

Expression of IL-23/Th17-related cytokines in basal cell carcinoma and in the response to medical treatments

Several immune-related markers have been implicated in basal cell carcinoma (BCC) pathogenesis. The BCC inflammatory infiltrate is dominated by Th2 cytokines, suggesting a specific state of immunosuppression. In contrast, regressing BCC are characterized by a Th1 immune response with IFN-γ promoting a tumor suppressive activity. IL-23/Th17-related cytokines, as interleukin (IL)-17, IL-23 and IL-22, play a significant role in cutaneous inflammatory diseases, but their involvement in skin carcinogenesis is controversial and is poorly investigated in BCC. In this study we investigated the expression of IFN-γ, IL-17, IL-23 and IL-22 cytokines in BCC at the protein and mRNA level and their modulation during imiquimod (IMQ) treatment or photodynamic therapy (PDT). IFN-γ, IL-17, IL-23 and IL-22 levels were evaluated by immunohistochemistry and quantitative Real Time PCR in 41 histopathologically-proven BCCs (28 superficial and 13 nodular) from 39 patients. All BCC samples were analyzed at baseline and 19 of 41 also during medical treatment (9 with IMQ 5% cream and 10 with MAL-PDT). Association between cytokines expression and clinico-pathological variables was evaluated. Higher levels of IFN-γ, IL-17, IL-23 and IL-22 were found in BCCs, mainly in the peritumoral infiltrate, compared to normal skin, with the expression being correlated to the severity of the inflammatory infiltrate. IFN-γ production was higher in superficial BCCs compared to nodular BCCs, while IL-17 was increased in nodular BCCs. A significant correlation was found between IFN-γ and IL-17 expression with both cytokines expressed by CD4+ and CD8+ T-cells. An increase of all cytokines occurred during the inflammatory phase induced by IMQ and at the early time point of PDT treatment, with significant evidence for IFN-γ, IL-23, and IL-22. Our results confirm the role of IFN-γ and support the involvement of IL-23/Th17-related cytokines in BCC pathogenesis and in the inflammatory response during IMQ and MAL-PDT treatments.

KIF3A and IL-4 are disease-specific biomarkers for psoriatic arthritis susceptibility

To date, the genes associated with Psoriatic Arthritis (PsA) are principally involved in inflammation, immune response and epidermal differentiation, without any information about the relationship between disease and bone metabolism genes. Our work was focused on 5q31 locus, which contains several genetic variants significantly associated with PsA. The study involved 1526 subjects (500 PsA, 426 PsV, 600 controls). The region was evaluated by selecting and genotyping the SNPs of interest by Real Time PCR and direct sequencing. The results were subjected to biostatistic and bioinformatic analysis. The case-control study highlighted a significant association between KIF3A/IL-4 and PsA, but not with PsV (Psoriasis Vulgaris) patients. In addition, the haplotype analysis revealed two haplotypes significantly associated with PsA susceptibility. The Linkage Disequilibrium (LD) study showed the presence of a specific block in high LD within 132,692,628-132,737,638 bp of 5q31, giving additional evidence of specific association of the 5q31 region in PsA patients. Moreover, KIF3A expression was assessed by immunohistochemistry assays which showed a marked and significant difference of KIF3A expression between pathological and normal tissues. Our analysis described KIF3A and IL-4 as novel susceptibility genes for PsA, suggesting a clear implication of bone metabolism genes in the disease etiopathogenesis.

Monitoring treatment response in patients affected by actinic keratosis: dermoscopic assessment and metalloproteinases evaluation after piroxicam 0.8% and sunfilter cream

Piroxicam (PXM), an enolic benzothiazine, inhibits the synthesis of cyclooxygenase-1 and -2, downregulates the production of prostaglandins and thromboxane, prevents polyamines production and suppresses metalloproteinases (MMPs) activities, the most prominent tumorigenesis-proteinases family (Campione et al., 2015; Jetter, Chandan, Wang, & Tsoukas, 2018). We recently studied the effects of long-term or short-term PXM treatment on actinic keratoses (AKs) and field of cancerization in immunocompetent subjects and organ transplant recipients (Babino et al., 2016; Campione, Diluvio, Paternò, & Chimenti, 2010; Garofalo et al., 2017). In this open-label trial, we described dermoscopic assessment of AK patients and evaluated the expression of MMP-1 and -2 at baseline and after treatment with PXM. Typical dermoscopic features seems to be associated with nonpigmented AKs: white-to-yellow surface scales, pink-to-red pseudonetwork, targetoid-like appearance, and rosette sign. Dermoscopic pattern of pigmented AKs are: multiple slate-gray-to-dark brown dots and globules, annular granular structures, uniform pigmented background and superficial, brown, broken-up pseudo-network (Fargnoli, Kostaki, Piccioni, Micantonio, & Peris, 2012; Zalaudek, & Argenziano, 2015). MMPs play a critical role in several processes including angiogenesis, cell proliferation, tumor invasion and metastasis (Polette, Nawrocki-Raby, Gilles, Clavel, & Birembaut, 2004). Greater MMP-2 expression was observed in high grade AKs (de Oliveira Poswar et al., 2015). Thirteen Caucasian patients affected by 30 AKs were enrolled (eight men and five women; age range: 61–78 years), after ethics committee approval. AKs were located on face, arms, dorsum of the hands, bald scalp, and upper back the upper limbs. PXM 0.8% and 50+ sun filters were applied twice daily for a period of 12 weeks. The clinical monitoring was performed by AKESA score at baseline, at intermediate visit and after 12-weeks-treatment (Campione et al., 2015). Dermoscopic images were obtained using a dermoscope (DermLite 3Gen LLC, Dana Point California) at 10-fold magnification and collected using DermLite foto equipment at 20to 50-fold magnification. A punch-biopsy was performed before and after therapy for morphological (Hematoxylin & Eosin staining [H&E]) and immunohistochemical evaluations. Immunoevaluation was based on the mean number of immunostaining cells per high power field, using an arbitrary score 0–3. Statistical analysis was developed using test for qualitative variables, and Students t-test and ANOVA for continuous variables. Differences were considered statistically significant for value of p < .05 (SPSS, version 12.0 Chicago, IL). After treatment, there was a highly significant clinical improvement in lesions based on the decrease of AKESA score (data not shown). The baseline dermoscopic features were red pseudo-network (90%), white/yellow areas and keratotic follicular openings (73.3%), strawberry pattern (63.3%), white-to-yellow scales (63.3%), telangiectasia (53.3%), annular granular structures and yellow dots surrounded by white rim (33.3%), gray pseudo-network (20%), rosette sign and glomerular vessels (16.6%). After treatment, white-to-yellow scales (13.3%), red pseudo-network (33.3%), strawberry pattern (16.6%), teleangiectasia (23.3%), annular granular structures (6.6%), white/yellow areas (16.6%), keratotic follicular openings (16.6%), yellow dots surrounded by white rim (3.3%), and rosette sign (0%) decreased. In two patient, gray pseudo-network and glomerular vessels, were unchanged (6.6%) (Figure 1). H&E staining confirmed typical morphological features of AK (hyperplasia, spongiosis, dermal elastosis) and restoration of normal skin architecture after PXM treatment. Also immunoevaluation of MMP-1 and MMP-2 showed a reduction of staining in treated-skin compared with baseline (Figure 2; p < .05). In most patients, a progressive reduction of AK dermoscopic criteria (rosette sign, annular granular structures, strawberry pattern, white/yellow areas, keratotic follicular openings, teleangectasia, glomerular vessels, gray pseudo-network, yellow dots surrounded by white rim) until their disappearance was observed after treatment. Red pseudo-network, white/yellow areas, and yellow scales still remaining in a few patients. Histological and immunoevaluation confirmed restoration of normal skin architecture and reduced levels of MMPs expression after treatment, confirming the role of this proteinases family in skin cancer. We report the dermoscopic assessment histological and immunoevaluation of AKs in patients treated by this novel non-ablative agent, confirming PMX as a promising therapy also from the dermoscopic and histological point of view. Received: 12 August 2018 Revised: 27 September 2018 Accepted: 6 October 2018 DOI: 10.1111/dth.12772