Silvia Fittipaldi

Nestin and WT1 expression in atheromathous plaque neovessels: association with vulnerability.

INTRODUCTION Neoangiogenesis is crucial for the progression and vulnerability of atheromasic lesions. Since adult vasa vasorum, which represent the neoangiogenetic burden of healthy arteries, constitutively express Nestin and Wilms Tumor (WT1), the aims of the present study are: i) to describe and quantify Nestin and WT1 in plaque neovessels; ii) to investigate the relationship between neovessel phenotype and plaque instability. METHODS We prospectively evaluated 49 consecutive carotid endarterectomy specimens. Histopathological characteristics were separately collected, particularly the intraplaque histological complications. Immunohistochemistry was carried out for CD34, Nestin and WT1; the density of positivity was evaluated for each marker. RT-PCR was performed to assess Nestin and WT1 mRNA levels on the first 10 plaques and on 10 control arteries. RESULTS Six (12.2%) plaques showed no neoangiogenesis. In the others, the mean immunohistochemical densities of CD34, Nestin, and WT1-positive structures were 41.88, 28.84 and 17.68/mm2. Among the CD34+ neovessels, 68% and 42% expressed Nestin and WT1 respectively, i.e., nearly 36% of the neovessels resulted to be Nestin+/WT1-. Furthermore, complicated plaques (n=30) showed significantly more CD34 and Nestin-positive vessels than uncomplicated plaques (n=13; P=0.045 and P=0.009), while WT1 was not increased (P=0.139). RT-PCR confirmed that WT1 gene expression was 3-fold lower than Nestin gene in plaques (p=0.001). CONCLUSIONS Plaque neoangiogenesis shows both a Nestin+/WT1- and a Nestin+/WT1+ phenotype. The Nestin+/WT1- neovessels are significantly more abundant in complicated (vulnerable) plaques. The identification of new transcription factors in plaque neoangiogenesis, and their possible regulation, can open new perspectives in the therapy of vulnerable plaques.

Inflammatory Mediators and Cerebral Embolism in Carotid Stenting: New Markers of Risk

Purpose To investigate serological predictors of risk for cerebral embolism after carotid artery stenting (CAS). Methods Twenty consecutive symptomatic and asymptomatic patients (13 men; mean age 74 years) with carotid artery stenosis undergoing standardized filter-protected CAS (Wallstent) were preoperatively evaluated to identify unstable plaque (duplex ultrasound), complicated aortic plaque (transesophageal echocardiography), and inflammatory status [high-sensitivity C-reactive protein (hs-CRP) and serum amyloid-A protein (SAA) serum levels]. Aortic arch type, carotid tortuosity, and complexity of the procedure were considered. Cerebral embolism was evaluated by comparing the number, volume, and side (ipsilateral and non-ipsilateral) of preoperative and postoperative cerebral lesions detected on diffusion-weighted resonance magnetic imaging (DW-MRI) and through light and scanning electron microscopy analysis of cerebral protection filters obtained from CAS. Results All CAS procedures were completed with no complications. All patients had a negative preoperative DW-MRI, but at least 1 asymptomatic cerebral lesion appeared on DW-MRI after the procedure in 18 (90%) patients. Female gender was associated with a higher number of cerebral lesions (18.2±10.9 vs. 8.3±8.8 for men, p=0.03). Carotid plaque morphology, supra-aortic vessel anatomy, and procedure complexity did not correlate with number or volume of new cerebral lesions. Complicated aortic plaque was associated with a higher volume of non-ipsilateral cerebral lesions than uncomplicated plaque (235.0±259.3 vs. 63.6±63.2 mm3, respectively; p=0.02). Hs-CRP ≥5 mg/L and SAA ≥10 mg/L were significantly associated with a higher number of new cerebral lesions [16.2±10.7 vs. 4.3±3.4 for hs-CRP <5 mg/L (p=0.02) and 14.8±10.3 vs. 2.8±3.4 for SAA <10 mg/L (p=0.006), respectively]. Hs-CRP ≥5 mg/L and SAA ≥10 mg/L also correlated with greater surface involvement by embolic materials in the protection filters at microscopic analysis [37.0% (5.1%) vs. 26.9% (2.5%) for hs-CRP <5 mg/L, p=0.004; 35.9% (13.5%) vs. 22.2% (6.9%) for SAA <10 mg/L, p=0.02]. Conclusion In addition to female gender and the presence of complicated aortic plaque, inflammatory status can be a predictor of cerebral embolism in CAS.

RUNX-1 and CD44 as markers of resident stem cell derivation in undifferentiated intimal sarcoma of pulmonary artery

Vasuri F, Resta L, Fittipaldi S, Malvi D & Pasquinelli G 
(2012) Histopathology 61, 737–743

In vitro alteration of physiological parameters do not hamper the growth of human multipotent vascular wall-mesenchymal stem cells

Background: Mesenchymal stem cells (MSCs) with multilineage potential and anti-inflammatory property can be isolated from different human tissues, representing promising candidates in regenerative medicine. Despite the common criteria of characterization, many factors contribute to MSC heterogeneity (i.e., tissue origin, coexistence of cell subsets at different stage of differentiation, epigenetic) and no standard methods have been approved to characterize MSCs in cell culture. Aim: The present study aimed to test whether MSCs resist adverse chemical and physical culture conditions, surviving MSC subpopulations are endowed with the stemness abilities; to characterize MMP expression in AAA-MSCs under the adverse experimental conditions. Methods and Results: MSCs enzymatically isolated from human abdominal aortic aneurysm (AAA-MSCs) were exposed to media acidification, hypoxia, starving, drying and hypothermia through the following strategies: (1) low-density seeding in closed flasks; (2) exposure to a chemical hypoxia inducer, cobalt chloride; (3) exposure to a dry environment with growing medium deprivation and culture at 4°C. None of these conditions affected MSC viability and stemness profile, as evidenced by NANOG, OCT-4, and SOX-2 mRNA expression in surviving cells. A significant MMP-9 decrease, especially when AAA-MSCs were exposed to hypothermia, was associated with stress resistant stem cells. Conclusions: AAA-MSCs survive to extremely adverse culture conditions, keeping their morphology and stemness features. Besides MMP-9 role in pathological tissue remodeling, this protease may be related to MSC survival. Future studies on MSCs derived from other tissues will be necessary to refine our culture protocol, which can represent an empirical method to demonstrate MSC stemness, with potential implications for their clinical use.

Facing the enigma of the vascular network in hepatocellular carcinomas in cirrhotic and non-cirrhotic livers.

Aims In this paper we aimed to analyse the typology and the phenotype of the different vascular modifications in human hepatocellular carcinomas (HCCs) with a new immunomorphological and gene expression approach. We also attempted to correlate these modifications with the histological parameters of tumour aggressiveness and the surrounding liver parenchyma. Methods Ninety-six HCCs (from 80 patients) were retrospectively enrolled, 46 occurring in non-cirrhotic livers, and 50 in livers transplanted for cirrhosis. Histopathological analysis, immunohistochemistry for CD34, Nestin and WT1 and RT-PCR for Nestin, transforming growth factor-β1 (TGFβ1) and insulin-like growth factor 1 (IGF1R) mRNA were performed in all nodules. Results By correlating the CD34 and Nestin immunoreactivity in HCC vasculature with the tumorous architecture, we identified four vascular patterns (named from ‘a’ to ‘d’). Each of them was characterised by different expressions of TGFβ1 and IGF1R mRNA. Pattern a showed CD34-positive/Nestin-negative sinusoids, and was prevalent in microtrabecular lesions. Pattern b showed similar morphology and architecture as pattern a, but with Nestin-positive sinusoids and a significant ‘boost’ in IGF1R and TGFβ1 mRNAs. In patterns c and d a progressive sinusoid loss and a gain of newly formed arterioles were seen. Notably, HCCs with pattern a arose more frequently in cirrhosis (p=0.024), and showed lower incidence of microvascular invasion (p=0.002) and infiltration (p=0.005) compared with HCCs with other patterns. Conclusions Although future studies are surely required, the identification of different vascular profiles in HCCs from cirrhotic and non-cirrhotic livers may help clarify the relationship between HCC progression and aggressiveness.

CoCl2 Administration to Vascular MSC Cultures as an In Vitro Hypoxic System to Study Stem Cell Survival and Angiogenesis.

Mesenchymal stem cells (MSCs) possess well-known reparative properties, among which the ability to form neovessels; in vivo, this characteristic is carried out both in a normal and in a pathological setting. Hypoxia, a condition common to many human diseases, is known to promote angiogenesis and to improve stem cell proliferation and differentiation. For this purpose, we provide an experimental protocol to test stem cell viability and angiogenesis under hypoxic conditions, comparing a vascular model of MSCs with stable cell lines. In order to avoid the use of expensive facilities, we propose the application of a chemical hypoxia inducer, cobalt chloride.

Diffuse calcifications protect carotid plaques regardless of the amount of neoangiogenesis and related histological complications.

Background. Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group showed that Nestin-positive intraplaque neovessels correlated with histological complications. The aim of the present work is to evaluate the relationship between neoangiogenesis, plaque morphology, and clinical instability of the plaque. Materials and Methods. Seventy-three patients (53 males and 20 females, mean age 71 years) were consecutively enrolled. Clinical data and 14 histological variables, including intraplaque hemorrhage and calcifications, were collected. Immunohistochemistry for CD34 and Nestin was performed. RT-PCR was performed to evaluate Nestin mRNA (including 5 healthy arteries as controls). Results. Diffusely calcified plaques (13/73) were found predominantly in females (P = 0.017), with a significantly lower incidence of symptoms (TIA/stroke (P = 0.019) than noncalcified plaques but with the same incidence of histological complications (P = 0.156)). Accordingly, calcified and noncalcified plaques showed similar mean densities of positivity for CD34 and Nestin. Nestin density, but not CD34, correlated with the occurrence of intraplaque hemorrhage. Conclusions. Plaques with massive calcifications show the same incidence of histological complications but without influencing symptomatology, especially in female patients, and regardless of the amount of neoangiogenesis. These results can be applied in a future presurgical identification of patients at major risk of developing symptoms.

Tissue miRNA 483-3p expression predicts tumor recurrence after surgical resection in histologically advanced hepatocellular carcinomas

The choice of surgical treatment for hepatocellular carcinoma (HCC) depends on several prognostic variables, among which histological features, like microvascular invasion and tumor grade, are well established. This study aims to identify the tissue miRNAs predictive of recurrence after liver resection in “histologically advanced” HCC. We selected 54 patients: 15 retrospective resected patients without recurrence (group A), 19 retrospective resected patients with HCC recurrence (group B), and 20 prospective patients (group C), with 4 recurrence cases. All selected HCC were “histologically advanced” (high Edmondson grade and/or presence of microvascular invasion). A wide spectrum of miRNAs was studied with TaqMan Human microRNA Arrays; qRT-PCR assays were used to validate results on selected miRNAs; immunohistochemistry for IGF2 was applied to study the mechanism of miR-483-3p. As a result, a significant differential expression between group A and B was found for 255 miRNAs. Among them we selected miR-483-3p and miR-548e (P<0.001). As a single variable (group C), HCC with miR-483-3p downregulation (mean fold increase 0.21) had 44.4% of recurrence cases; HCC with miR-483-3p upregulation (mean fold increase 5.94) showed no recurrence cases (P=0.011). At immunohistochemistry (group C), the HCC with loss of cytoplasmic IGF2 expression showed a down-regulation of miR-483-3p (fold increase 0.57). In conclusion, in patients with “histologically advanced” HCC, the analysis of specific tissue miRNAs (particularly miR-483-3p) could help identify the recurrence risk and choose which treatment algorithm to implement (follow-up, resection or transplantation). This could have an important impact on patient survival and transplantation outcome, improving organ allocation.

From large to small: The immunohistochemical panel in the diagnosis of early hepatocellular carcinoma

The aims of this study were to: validate the use of the immunohistochemical (IHC) markers glutamine synthetase (GS), glypican‐3 (GPC3), heat shock protein‐70 (HSP70) and enhancer of zeste homologue 2 (EZH2) in liver biopsies for the differential diagnosis between small hepatocellular carcinoma (HCC) and non‐neoplastic liver nodules, with special attention to <10‐mm nodules; and assess the actual sensitivity and specificity of the single markers, and their combination, in needle biopsies.

miRNA Signature of Hepatocellular Carcinoma Vascularization: How the Controls Can Influence the Signature

BackgroundmiRNA deregulation and vascular modifications constitute promising predictors in the study of hepatocellular carcinoma (HCC). In the literature, the relative miRNA abundance in HCC is usually determined using as control non-matched tumoral tissue, healthy liver, or cirrhotic liver. However, a common standard RNA control for the normalization toward the tissue gene expression was not settled yet.AimTo assess the differences existing in the quantitative miRNA gene expression in HCC on tissue according to two different liver controls.MethodsA wide array of miRNAs was analyzed on 22 HCCs arisen in cirrhotic and non-cirrhotic livers by means of microfluidic cards. Control samples included total RNA extracted from healthy and cirrhotic livers. Immunohistochemistry for CD34 and Nestin was performed to assess the pattern of intratumoral vascular modifications.ResultsSix miRNAs were deregulated in HCCs using either controls: miR-532, miR-34a, miR-93, miR-149#, miR-7f-2#, and miR-30a-5p. Notably, the miRNA expression changed significantly between HCCs arisen in cirrhotic and non-cirrhotic livers, according to the control used for normalization. Different miRNA profiles were found also in HCCs with different vascular patterns, according to the control used for normalization.ConclusionsOur data confirm that the choice of the methodology, and particularly the control used for normalization, represents the main concern in miRNA evaluation, particularly in a heterogeneous model such as liver pathology. Still we observed the deregulation of some common miRNAs as promising in HCC cancerogenesis and progression. A standardized control will be a crucial achievement to compare miRNA expression among different laboratories.