Gianfranco Tajana

Microarray analysis: a novel research tool for cardiovascular scientists and physicians

The massive increase in information on the human DNA sequence and the development of new technologies will have a profound impact on the diagnosis and treatment of cardiovascular diseases. The microarray is a micro-hybridisation based assay. The filter, called microchip or chip, is a special kind of membrane in which are spotted several thousands of oligonucleotides of cDNA fragments coding for known genes or expressed sequence tags. The resulting hybridisation signal on the chip is analysed by a fluorescent scanner and processed with a software package utilising the information on the oligonucleotide or cDNA map of the chip to generate a list of relative gene expression. Microarray technology can be used for many different purposes, most prominently to measure differential gene expression, variations in gene sequence (by analysing the genome of mutant phenotypes), or more recently, the entire binding site for transcription factors. Measurements of gene expression have the advantage of providing all available sequence information for any given experimental design and data interpretation in pursuit of biological understanding. This research tool will contribute to radically changing our understanding of cardiovascular diseases.

Involvement of β3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function

The β3-adrenoreceptor plays a major role in lipolysis but the role and distribution of β3-receptors in other specific sites have not been extensively studied. β3-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, β3-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO–cGMP signal transduction pathway. Here we show that β3-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective β3-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific β3-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal β3-receptor-mediated vasorelaxant tone and that β3-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that β3-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.

Evidence that protease activated receptor 2 expression is enhanced in human coronary atherosclerotic lesions.

Aim: To investigate protease activated receptor 2 (PAR-2) expression in human coronary atherosclerotic lesions because PAR-2 is involved in the modulation of inflammatory events and vascular function. Methods: An immunohistochemical analysis was performed on serial arterial sections, using the following antibodies: MDA2, a murine monoclonal antibody against malondialdehyde lysine epitopes of oxidised low density lipoprotein (oxLDL); HAM-56, a monoclonal antibody against human macrophages/foam cells; B5, a rabbit polyclonal antibody against PAR-2; and SAM11, a mouse monoclonal antibody against human PAR-2. Sections containing at least one lesion showing substantial immunostaining were counted as positive, and results were expressed as per cent of all sections of the same artery. Results: PAR-2 expression was enhanced in human coronary atherosclerotic lesions. This phenomenon correlated with an increase in oxLDL epitopes in the coronary artery. Conclusion: This study shows for the first time that PAR-2 expression is enhanced in human coronary atherosclerotic lesions, and suggests that PAR-2 dependent cellular trafficking may be one of the regulatory signalling responses to vascular injury. Further pharmacological studies will establish whether modulation (and in which direction) of PAR-2 represents a possible therapeutic target for controlling the vascular response to injury.

c-Myc Oncoprotein: Cell Cycle-Related Events and New Therapeutic Challenges in Cancer and Cardiovascular Diseases

Advanced stages of both cancer and atherosclerosis are characterized by a local increase in tissue mass that may be hard to control. This increase in tissue mass can be attributed to oxidation-sensitive modification of cell cycle-related events, including cellular proliferation, differentiation, and apoptosis, which could be secondary to alteration in the activity of tumor suppressor gene and oncogene products. The oncogene c-Myc has classically been considered to be involved in carcinogenesis and has more recently been implicated in both endothelial dysfunction and atherogenesis as well. Consequently, inhibition of c-Myc-dependent signaling has become a novel therapeutic opportunity and challenge in atherosclerosis and other cardiovascular diseases. Antioxidant strategies, RNA synthesis inhibitors such as mithramycin, and gene therapeutic approaches with antisense oligonucleotides against c-Myc are some of the promising strategies. In general, the increased biologic understanding of the participation of cell cycle events and targeting these events may enable to attenuate or prevent some of the complications of vascular and neoplastic diseases.

A New Biopsy Technique to Investigate Peyronie’s Disease Associated Histologic Alterations: Results with Two Different Forms of Therapy

OBJECTIVES Peyronies disease is the most frequent cause of penile curvature and occurs particularly in middle-age patients. The best technique for penile biopsy, in the evaluation of albuginea and cavernous tissue, has not been delineated yet. We present a new technique of penile biopsy, useful in the study of Peyronies plaque, fibrosis and erectile dysfunction or any other pathological condition of the penis requiring a biopsy. METHODS A treatment group (A) of 380 patients underwent Extra Shock Waves Treatment (ESWT) three times a week for 20 minutes, followed by a complete cycle of 12 injections of verapamil (10mg), every two weeks for six months. A control group (B) of 92 patients underwent verapamil injections alone. Three months after the end of the treatment, each patient underwent penile biopsy performed with Acu-Punch (Acuderm Inc.), a biopsy-punch armed with a well-sharpened rotating cylindrical blade, first used by dermatologists for cutaneous lesions. RESULTS A reduction of the plaque volume was found in 260/380 patients (68.4%) of group A and in 28/92 (30.4%) of group B; painful erection weaned off in 312/340 patients of group A (91.7%) and in 36/82 patients (43.9%) of group B. In all 472 patients an excellent specimen was obtained and both the tunica albuginea and the cavernous tissue were easily identified. In the 260 cases, in which the Extra Shock Waves Treatment was successful, scanning and transmission electron microscopy demonstrated a reduction in packing and clumping of the collagen fibers. CONCLUSIONS This new technique of penile biopsy with Acu-Punch can replace surgical biopsies when a surgical operation is not indicated. Such a low-invasive technique could be performed in all cases of Peyronies disease and allows a more extensive use of microscopic analysis in the study of Peyronies disease.

Androgens and morphologic remodeling at penile and cardiovascular levels: a common piece in complicated puzzles?

CONTEXT Epidemiologic data demonstrate a protective role by normal androgen levels on cardiovascular health and erectile function. Low androgen levels are associated with erectile dysfunction and increased risk of cardiovascular diseases. Both conditions recognize as anatomic substrate a pathologic structural remodeling. Direct androgen effects on male external genitalia, vascular wall, and myocardium have been reported. OBJECTIVE To review current knowledge about androgen-dependent molecular signaling pathways and cellular events within penile and cardiovascular tissues involved in the homeostatic control of morphologic tissue properties and in the development of structural remodeling in presence of normal and low androgen levels, respectively. EVIDENCE ACQUISITION A literature search was performed in November 2008 using the commercially available Medline online engine search to retrieve studies (from 1998 to 2008) on the mechanisms mediating the role of androgens on penile and cardiovascular morphologic homeostasis and remodeling. A combination of the following medical subject headings was used: androgens, hypogonadism, vessel tissue architecture, remodeling, cardiovascular system, and penis. EVIDENCE SYNTHESIS Androgens exert direct beneficial effects on both cardiovascular and penile tissues. Endothelial cells and smooth-muscle cells are the main cellular targets for direct androgen effects in both tissues and are involved in pathologic remodeling in hypogonadal models. At vascular level, androgens promote endothelial cell survival, reduce endothelial expression of proinflammatory markers, and inhibit proliferation and intimal migration of vascular smooth-muscle cells. At penile level, low androgen levels are associated with apoptosis of endothelial cells and smooth-muscle cells. Moreover, low androgen levels impair proliferation, migration, and homing of endothelial progenitor cells as well as myogenic differentiation of mesenchymal progenitor cells. CONCLUSIONS Normal androgen levels promote vascular and penile homeostasis by direct mechanisms mainly involving endothelial cells and smooth-muscle cells. Low androgen levels are associated with impairments of such mechanisms, leading to pathologic structural remodeling.

A standardized procedure for using human corpus cavernosum strips to evaluate drug activity

The main problem of using human corpus cavernosum (HCC) tissue to perform bioassay is linked to its limited availability further complicated by the heterogeneous source of the tissues used. Here, we show that gender reassignment is a reliable source of human tissue without major ethical problems. Indeed, the entire corpus cavernosum is obtained from the surgery procedure, which allows creating a standardized procedure to prepare HCC strip. In addition, human tissue, if kept in the fridge in the condition described, does not loose its ability to contract to phenylephrine (PE; alpha agonist), angiotensin II (AG II) and KCl up to 4 days. Furthermore, once contracted with PE, HCC relaxes to acetylcholine (endothelium-dependent mechanism); sodium nitroprusside (endothelium-independent mechanism); cromakalim (CRK), a K(ATP) channel opener; or alprostadil, a synthetic PGE2 (ALPR). In conclusion, we have standardized a procedure that allows the use of HCC strips to evaluate drug activity and/or to study pathophysiological mechanisms with an intact functional human tissue up to 4 days from the surgery procedure.

I.S.Mu.L.T - Rotator cuff tears guidelines

Despite the high level achieved in the field of shoulder surgery, a global consensus on rotator cuff tears management is lacking. This work is divided into two main sessions: in the first, we set questions about hot topics involved in the rotator cuff tears, from the etiopathogenesis to the surgical treatment. In the second, we answered these questions by mentioning Evidence Based Medicine. The aim of the present work is to provide easily accessible guidelines: they could be considered as recommendations for a good clinical practice developed through a process of systematic review of the literature and expert opinion, in order to improve the quality of care and rationalize the use of resources.

The immunomodulatory protein SV-IV protects serum-deprived cells against apoptosis but not against G0/G1 arrest: possible implications for the survival of implanting embryo.

Serum deprivation induced in human lymphoblastoid Raji cells oxidative stress‐associated apoptotic death and G0/G1 cell cycle arrest. Addition into culture medium of the immunomodulatory protein Seminal vesicle protein 4 (SV‐IV) protected these cells against apoptosis but not against cycle arrest. The antiapoptotic activity was related to: (1) decrease of endocellular reactive Oxygen species (ROS) (2) increase of mRNAs encoding anti‐oxidant enzymes (catalase, G6PD) and antiapoptotic proteins (survivin, cox‐1, Hsp70, c‐Fos); (3) decrease of mRNAs encoding proapoptotic proteins (c‐myc, Bax, caspase‐3, Apaf‐1). The biochemical changes underlaying these effects were probably induced by a protein tyrosine kinase (PTK) activity triggered by the binding of SV‐IV to its putative plasma membrane receptors. The ineffectiveness of SV‐IV to abrogate the cycle arrest was accounted for by its downregulating effects on D1,3/E G1‐cyclins and CdK2/4 gene expression, ppRb/pRb ratio, and intracellular ROS concentration. In conclusion, these experiments: (1) prove that SV‐IV acts as a cell survival factor; (2) suggest the involvement of a PTK in SV‐IV signaling; (3) point to cell cycle‐linked enzyme inhibition as responsible for cycle arrest; (4) provide a model to dissect the cycle arrest and apoptosis induced by serum withdrawal; (5) imply a possible role of SV‐IV in the survival of hemiallogenic implanting embryos. J. Cell. Physiol. 212:610–625, 2007.

Glycoxidation of Low-Density Lipoprotein Increases TUNEL Positivity and CPP32 Activation in Human Coronary Cells

Abstract: Apoptosis of arterial cells induced by oxidized low‐density lipoprotein (oxLDL) is thought to contribute to the progression of vascular dysfunction and atherogenesis. It is well established that diabetes mellitus is accompanied by both glycosylation and oxidation of LDL (glc‐oxLDL), but the biological effects of these modified lipoproteins are poorly understood. We demonstrate here for the first time that glc‐oxLDL increases TUNEL positivity and caspase‐3 activation (by Western blot and immunocytochemistry) of human coronary smooth muscle cells. Overall, these effects induced by glc‐oxLDL were greater than those achieved with oxLDL. Thus, glc‐oxLDL activated downstream apoptotic signaling. This may influence the evolution of atherogenesis and vascular complications in diabetes.