Identification of microRNAs 758 and 33b as potential modulators of ABCA1 expression in human atherosclerotic plaques.

BACKGROUND AND AIM: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters A1 and G1 are the main transporters involved in macrophage cholesterol efflux. The understanding of the molecular mechanism(s) of their regulation in atherosclerosis is crucial for potential therapeutic approaches. Preclinical studies support a role for microRNAs in the posttranscriptional regulation of these transporters; however, no evidence is still available on human atherosclerosis. Thus, the aim of this study was to investigate the modulation of the ABCA1 and ABCG1 pathway in human atherosclerotic plaques and microRNA involvement in its modulation. METHODS AND RESULTS: Thirty-one human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy for high-grade (>70%) vessel stenosis, and divided into normocholesterolemic (n = 15) and hypercholesterolemic groups (n = 16) according to the presence/absence of hypercholesterolemia. Both ABCA1 and ABCG1 messenger RNAs (mRNAs) were significantly upregulated in carotid plaques from hypercholesterolemic patients as assessed by real-time polymerase chain reaction (RT-PCR). Despite this result, no difference was found at the protein levels analyzed by Western blot, thus suggesting a strong posttranscriptional modulation. MicroRNA microarray and subsequent validation by RT-PCR showed a significant upregulation of ABCA1-linked miR-758 and miR-33b in plaques from hypercholesterolemic patients. CONCLUSION: We provide evidence of a strong posttranscriptional regulation of ABCA1 and ABCG1 expression in human atherosclerotic plaques from hypercholesterolemic patients. This effect is potentially due to the concomitant increase of miR-33b and miR-758, two well-established regulators of ABCA1 and ABCG1 expression. The identification of miR-33b and miR-758 as putative key regulators of ABCA1 protein expression within human atherosclerotic plaques provides further data for the realization of new anti-atherosclerotic drugs with specific targets based on anti-miRNA technologies.

Factors associated with apoptosis in symptomatic and asymptomatic carotid atherosclerotic plaques.

The aim of this study was to investigate the differences that are present between apoptosis in symptomatic (with symptoms of cerebral ischemic attack) and asymptomatic carotid atherosclerotic plaques. The apoptotic process in macrophages and smooth muscle cells was evaluated. Cellular markers and products of immune cells in symptomatic and asymptomatic atherosclerotic plaque and endoarterectomy specimen were analyzed by immunohistochemistry. No statistically significant differences were present regarding the mean SMC actin-positive area. Using double staining of alpha-smooth muscle actin and TUNEL techniques, the number of smooth muscle cells in apoptosis was statistically higher in symptomatic plaque as compared with asymptomatic plaque. Statistically significant differences (p=0.009) were also found in the CD45-positive cells in the inflammatory infiltrate. The CD68-positive macrophages showed statistically significant differences (p=0.0001). Similarly, the double staining with CD68 and TUNEL revealed that apoptotic macrophages were mainly present in asymptomatic plaques rather than symptomatic plaques. Statistically significant differences (p<0.001) were found in the Bcl-2 expression, with higher values in asymptomatic plaques. Our data showed that the increase of the inflammatory cells contributes to plaque instability and that death due to apoptosis of smooth muscle cells in symptomatic plaques could contribute to their destabilization and explains their tendency to fracture.

Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E(2)-dependent plaque instability.

BACKGROUND: Studies have implicated a role for prostaglandin (PG) E(2)-dependent matrix metalloproteinase (MMP) biosynthesis in the rupture of atherosclerotic plaque. Cyclooxygenase-2 (COX-2) and PGE synthase (PGES) are coregulated in nucleated cells by inflammatory stimuli. The aim of this study was to characterize the expression of COX-2 and PGES in carotid plaques and to correlate it with the extent of inflammatory infiltration and MMP activity and with clinical features of patients' presentation. METHODS AND RESULTS: Plaques were obtained from 50 patients undergoing carotid endarterectomy and divided into 2 groups (symptomatic and asymptomatic) according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, PGES, MMP-2, and MMP-9 by immunocytochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunocytochemistry was used to identify CD68+ macrophages, CD3+ T lymphocytes, and HLA-DR+ cells. The percentage of macrophage-rich areas was larger (P<0.0001) in symptomatic plaques. COX-2, PGES, and MMPs were detected in all specimens; enzyme concentration, however, was significantly higher in symptomatic plaques. COX-2, PGES, and MMPs were especially noted in shoulders of symptomatic plaques, colocalizing with HLA-DR+ macrophages. All symptomatic plaques contained activated forms of MMPs. Finally, inhibition of COX-2 by NS-398 was accompanied by decreased production of MMPs that was reversed by PGE(2). CONCLUSIONS: This study demonstrates the colocalization of COX-2 and PGES in symptomatic lesions and provides evidence that synthesis of COX-2 and PGES by activated macrophages is associated with acute ischemic syndromes, possibly through metalloproteinase-induced plaque rupture.

Increased systemic oxidative stress after elective endarterectomy: relation to vascular healing and remodeling.

It has been reported that systemic and local redox state may have an important role in the functional and organic changes characterizing the process of vascular response to injury. Carotid endarterectomy to remove atherosclerotic plaque is followed by a long lasting healing and remodeling process that can be carefully followed over time with noninvasive ultrasonography. Plasma vitamin C concentration and native LDL (n-LDL) content in lipid peroxides, vitamin E, beta-carotene, and lycopene as well as LDL susceptibility to peroxidation were assessed in 45 patients undergoing elective endarterectomy for internal carotid stenosis, at baseline, 24 hours, 3 and 15 days, and 1 month after surgery. Serial duplex scans were performed in all patients postoperatively and 3, 6, and 12 months. The changes in far wall thickness (FW) and % renarrowing from postoperatively to 12 months were used as remodeling indices. Plasma antioxidant vitamins and lag-phase showed a sharp and significant decrease during the first 24-hours after surgery remaining unchanged until the third day, whereas, an opposite trend was evidenced for n-LDL content in lipid peroxides and serum ceruloplasmin. After the third day all the parameters returned progressively to baseline within one month from endarterectomy. Interestingly, the n-LDL lipid peroxide content, the serum ceruloplasmin and the plasma vitamin C concentration, measured at 24 and 3 days from surgery, were significantly associated to the change in % renarrowing from postoperatively to 12 months. The higher the LDL content in lipid peroxides, the higher the serum level of ceruloplasmin, the lower the plasma content in vitamin C and the higher the % of vessel renarrowing. In conclusion, carotid endarterectomy with atherosclerotic plaque removal is associated with an acute and prolonged increase in systemic oxidative stress that influences vascular healing and late luminal loss.

[Aneurysms of the subclavian artery: clinical experience]. / Gli aneurismi dell'arteria succlavia: esperienza clinica.

The aneurysm of the subclavian artery is not common. The authors present three cases of aneurysms of the subclavian artery of which two cases already were in phase of complication for rupture. Two Patients underwent intervention of by-pass with Dacron prosthesis: between common carotid and axillary artery in one and between common carotid and distal head of the subclavian artery in the other. In a Patient with post-traumatic pseudoaneurysm of the subclavian artery was not possible to perform any intervention of direct reconstruction because of the precarious clinical conditions due to the hemorrhage for rupture and we performed only a resection of the aneurysm with binding of the subclavian artery. A Patient died two months after the operation for respiratory failure due to bronchial pneumonia; two Patients are still living, one with a moderate motor deficit of the upper limb. The rupture of the arteriosclerotic aneurysms of the subclavian artery is not frequent, but the gravity of this complication recommends the surgical treatment also in the asymptomatic cases if the clinical conditions of the Patient allow it.

[Acute thrombosis of the infrarenal aorta]. / Trombosi acuta dell'aorta infrarenale.

The Authors report three cases of patients operated for acute thrombotic aortic occlusion. All the three patients were treated with an aortic transabdominal approach, thromboendarteriectomy and aorto-femoral by-pass. In two patients the result was satisfying, one with a late and partial recovery of the motility of the inferior limbs. One patient died for heart failure in eighth postoperative day. The Authors believe that this unsatisfying result was caused by delay in the surgical treatment (almost 30 hours).

Glutathione-related antioxidant defenses in human atherosclerotic plaques.

BACKGROUND: Oxidative stress, resulting from an antioxidant/prooxidant imbalance, seems to be crucial in atherogenesis. Recent evidence has emerged, however, of a surprisingly high content of low-molecular-weight antioxidants in human atherosclerotic plaques, although other antioxidant systems have not been investigated in these lesions. METHODS AND RESULTS: We studied glutathione-related antioxidant defenses (which play a key role in tissue antioxidant protection) in carotid atherosclerotic plaques of 13 patients subjected to endarterectomy and in normal internal mammary arteries of 13 patients undergoing coronary artery bypass surgery. Selenium-dependent glutathione peroxidase activity was undetectable in the plaques of 7 patients; the other 6 patients with plaques showed a mean enzymatic activity approximately 3.5-fold lower than that of mammary arteries. Glutathione reductase activity was also markedly lower in the plaques than in the arteries. Glutathione transferase instead had comparable activity in the two tissues. Remarkably, 5 of the 7 patients with an undetectable selenium-dependent glutathione peroxidase activity but none of the 6 with a detectable one were characterized by multivascular atherosclerotic involvement (3 patients) or stenosis of the contralateral carotid artery (2 patients). CONCLUSIONS: A weak glutathione-related enzymatic antioxidant shield is present in human atherosclerotic lesions. Although the cause of this phenomenon remains to be determined, the present data suggest that a specific antioxidant/prooxidant imbalance operative in the vascular wall may be involved in atherogenic processes in humans.

[Unusual pathology of the spleen]. / Patologie non frequenti della milza.

The Authors report 7 cases of unusual spleen pathology represented by 2 real cysts, 1 pseudocyst, 1 hydatid cyst, 2 abscesses, 1 splenic artery aneurysm. Pathophysiological hypotheses are examined, and clinical and diagnostic considerations are stressed, evaluating particularly anatomo-histologic characteristics and the differential diagnosis through non invasive imaging techniques. All patients were subjected to splenectomy via laparotomy, except for the case of real cyst where an inferior hemisplenectomy was performed. In the case of splenic artery aneurysm, an urgent aneurysmectomy was required together with splenectomy, considering the young age of the patient (29 years old), and the type of aneurysm presenting in a double rupture phase. Reoperation was necessary in only one patient to drain an hematic abscess. After a 9 year follow up all the patients are well.

[Personal experience in the treatment of carcinoma of the head of the pancreas]. / Esperienza personale nel trattamento del carcinoma della testa del pancreas.

The Authors report 195 cases of carcinoma of the head of the pancreas observed in the period 1983-1995. Only 94 (48%) of these patients were operated: in 32 cases an exploratory laparotomy was performed, while in 36 a bilio-digestive bypass, associated in 2 patients with a gastro-jejunal bypass was assured. In 26 patients a resection was performed: in 12 a Whipple procedure, in 6 a total duodenopancreatectomy, in 8 a pylorus preserving procedure (Traverso-Longmire). The mean survival was 2 months after laparotomy, 6 months after bilio-digestive bypass, 20 months after Whipple procedure, 20 months after total duodenopancreatectomy, 23 months after a pylorus preserving procedure (Traverso-Longmire). The Authors believe that the Whipple procedure is the treatment of choice for the carcinoma of the head of the pancreas. Only in cases of suspected multifocality or when there is a severe nodal involvement it is advisable to perform a total duodenopancreatectomy.

[Clinical considerations of cases of biliary ileus]. / Considerazioni cliniche su alcuni casi di ileo biliare.

The authors report 6 cases of gallstone ileus due to cholecystoduodenal fistula. In 4 patients a simple enterolithotomy was performed, while in one case the enterolithotomy was associated to cholecystectomy and duodenal suture; in the last patient it was associated to cholecystectomy and gastric resection. Only one death was registered among the cases treated with enterolithotomy. The authors believe that the severity of the pathology, generally combined with other important diseases, needs an eclectic treatment depending on the patient status.

[Gynecomastia]. / Ginecomastia.

Gynaecomastia is a benign enlargement of the male breast commonly occurring in healthy adolescent boys and in adults (50 to 80 years old). It is a source of cosmetic and psychologic problems. The secondary forms usually require no therapy other than the removal of any identified inciting cause. Most of the idiopathic forms last only a few months and then gradually disappear. In secondary gynaecomastia and idiopathic forms present for more than 12 months a medical hormonal treatment often fails and the only therapy is surgery consisting in a subtotal subcutaneous mastectomy. In the last years the surgical treatment is often completed with suction lipectomy: this procedure reduces the surgical traumatism. The authors present their experience concerning 47 patients: 27 were surgical treated with subcutaneous subtotal mastectomy and 18 had an abjunctive liposuction treatment with good results.

[Skeletization of the inferior mesenteric artery in colorectal surgery. Current considerations]. / La scheletrizzazione dell'arteria mesenterica inferiore nella chirurgia colo-rettale. Considerazioni attuali.

Anastomotic dehiscence after colon resection is the most frequent complication in colon surgery and the main cause of post-operative death. In the light of anatomical peculiarities of the blood supply to the rectum, it would appear that in atherosclerotic patients with impairment of hypogastric arteries (80% in authors' series out of 200 atherosclerotic subjects) inferior mesenteric artery ligature, determining vascularization of the rectal ampulla by the distal vessels alone, results in an insufficient supply in case of colo-rectal anastomosis. A series of 15 cases of cancer of the left and sigmoid colon, treated with left hemicolectomy, preservation and peeling of the inferior mesenteric artery, is reported. In the follow-up ranging from 6 months to 5 years, no anastomotic dehiscence was observed and only one case (7.5%) presented hepatic recurrence after two years. The other patients are all alive and disease free.

[Injury of the popliteal artery & the popliteal branches: analysis of 31 consecutive cases]. / I traumi dell'arteria poplitea e dei rami infrapoplitei: analisi di 31 casi consecutivi.

In complex traumas of lower limbs it is fundamental the reducing of the time of ischemia to reduce the number of failures in repairing surgery and the percentage of the demolishing of necessity surgery. The experience of the authors is based on 31 complex traumas of lower limbs with distal vascular injuries to Hunter's canal, which were associated to bone, nervous and muscular lesions. In total we are treated 37 vascular injuries by interposition of venous autografts in 37 cases, lateral pacth in one case ant suture T-T in two cases. The protection of the reconstructed vases, in case of a concomitant loss of substance, was entrused to microvascular flaps for 7 times latissimus dorsi, iliac crest and fascio-cutaneous for 5 times posterior reversed with distal baset and for three times antero-lateral, which in three cases of them needed successively a dermoepidermic grafts. The bone injuries, which were treated by external fixation, intramedullary nailing, plate and screws, just in two cases became worse in pseudoarthroses and just in one in osteomyelitis. The reparation of nervous injuries had bad results just in two cases. The percentage of saving of the limb it was about 83.9%.

Carotid bifurcation stenosis: a comparative study between MR angiography and duplex scanning with respect to digital subtraction angiography.

PURPOSE: This study compared sensitivity, specificity and diagnostic accuracy of Echoduplex and Magnetic Resonance Angiography (MRA) in the evaluation of carotid bifurcation stenosis. MATERIAL AND METHODS: Twenty-five patients with clinical signs suggestive for cerebrovascular insufficiency (CVI) were studied with Duplex scan, MRA and Digital Subtraction angiography (DSA). Gold standard was the angiographic examination. RESULTS: on equal value of sensitivity (80.5%), MRA showed 96% specificity versus 81% of Duplex scan, 89.4% diagnostic accuracy versus 80.9% for Duplex scan. As for stenosis over 31%, the value of sensitivity did not change i.e., 80.5%; MRA showed 100% specificity versus 97.1% for Duplex, 91.2% diagnostic accuracy versus 89.1% respectively. These values compared by the test for categorial analysis and correspondence analysis (p < 0.05) did not indicate any statistically significant difference. DISCUSSION: on the basis of our experience and as shown by current literature [2, 12] we can state that both MR-angiography and Duplex scan fail in quantifying correctly carotid stenosis with consequent over- and underestimation [3, 4]. However, they can be considered effective diagnostic procedures in a screening program [12, 16]; they are accurate, safe and accepted by the population. CONCLUSIONS: on the basis of the cost in planning the screening of a population at risk for CVI, Duplex scanning is still to be considered the elective procedure.

Antiproliferative effect of desferrioxamine on vascular smooth muscle cells in vitro and in vivo.

Vascular smooth muscle cell (VSMC) growth is a primary component of accelerated and spontaneous atherosclerosis. Previous studies have shown that iron may be involved in the control of enzymatic activities that modulate DNA synthesis in human cells. In this study the effects of the iron chelator desferrioxamine on in vitro and in vivo VSMC proliferation were tested. Rat VSMCs in culture and a rabbit model of carotid artery balloon injury were used. Desferrioxamine showed a significant inhibitory effect on [3H]thymidine incorporation in cell cultures that was antagonized by iron supplementation. Desferrioxamine also provided effective preventive myointimal VSMC proliferation as assessed by bromodeoxyuridine labeling and morphometric analysis of endoluminal stenosis. These experiments suggested that iron may be involved in the control of VSMC proliferation and that desferrioxamine may have a role in preventing VSMC growth and myointimal proliferative lesions.

[Carotid thromboendarterectomy. The authors' personal experience]. / La tromboendoarteriectomia carotidea. Esperienza personale.

Carotid endarterectomy is the most frequently employed surgical intervention in the treatment of strokes due to atherosclerosis obstruction of the blood flow in extracranial carotid district. The presence of cerebrovascular clinical symptoms is accepted indication for surgical treatment. For the patients without symptoms there is, however, not yet a complete agreement on the treatment modalities. The authors believe that, due to the low morbidity and mortality of carotid endarterectomy, a surgical approach can be used in patients in which is present a degree of stenosis > 75% and, moreover, in patients where the degree of stenosis is > 50%, but in which the atherosclerotic plaque is non homogeneous due to the presence of ulceration or hemorrhage. Non invasive ultrasonic techniques constitute the mainstay of the diagnostic procedures for carotid artery disease and in recent years they have led to improved sensitivity and ability in differentiating between the operable carotid stenoses and the inoperable obstructions. With these techniques it is possible to arrive to a 90% diagnostic accuracy. Also the recently introduced angio-RM has comparable results, but the high costs do not make it possible to use it in screening procedures. Many doubts are present regarding the possibility of evaluating the occurrence of neurologic deficits during the surgical procedures. Local anaesthetic techniques are somewhat popular among vascular surgeons in that they allow an immediate evaluation of the neurologic activity of the patients during surgical treatment. However reports have been published in the literature of neurologic deficits arising after the period of test occlusion.

The treadmill test in the assessment of peripheral arterial occlusive disease.

Results of a study using the treadmill test on 32 patients with peripheral Fontaine's stage II arterial disease are described. The aim of the study was to evaluate the utility of the test in assessing useful information concerning the site of the lesion. Patients were divided in three classes depending on the arterial segment involved (iliac-femoral, femoro-popliteal, associated lesions) as resulted from the velocimetric and angiographic evaluation. Ankle pressure and WI, at rest and after stress test, PFWD and RT were evaluated. The authors conclude that data achieved with the dynamic test are related to the arterial involvement, allowing to differentiate obstructive and stenotic lesions, but do not give information concerning the site of the lesion. Nevertheless it is a valuable means of highlighting arterial lesions well compensated at rest and with normal pressure values.

[Autonomous adenoma of the thyroid]. / L'adenoma autonomo della tiroide.

Toxic goitre often referred to in the literature as "autonomous" adenoma or nodule actually signifies a number of different pathologies that are characterised by autonomy and hyperfunction so that it is now thought more appropriate to call it an autonomous follicle. Clinical features and the hormone profile suggest the diagnosis which is confirmed by scintiscan with curve as well as dynamic inhibition and stimulation tests using TRH, procedures that reveal both latent hyperfunction and autonomy. The most common current therapeutic approach is surgical since this provides a complete cure while leaving the surrounding thyroid tissue undisturbed.