Paola Lorenzoni

Antiangiogenic VEGF Isoform in Inflammatory Myopathies

Objective. To investigate expression of vascular endothelial growth factor (VEGF) antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM) and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis) and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

Culture of bovine thoracic duct endothelial cells

Dear Editor: In vitro studies have led to substantial advances in our understanding of the biology and physiologic role of blood vessel endothehum. The study of lymphatic endothelial cells (LEC) has been delayed due to the technical problems of isolation. LEC have been successfully isolated from bovine mesenteric lymphatic collectors (3-5,8), from canine thoracic duct (2), from a retroperitoneal recurrent lymphangioma (7), and more recently, from rat thoracic duct (1). LEC have also been isolated from human thoracic duct but these cells failed to grow in culture (2). The number of lymphatic endothelial cells that can be obtained from bovine mesenteric lymphatics and from the thoracic duct of dog and rat is, however, quite low due to the small size of these vessels, and insufficient for most biochemical studies. Here we report a reliable and easily reproducible technique for the isolation of a relatively large number of LEC from the main bovine lymphatic vessel, the thoracic duct. They may be utilized for biochemical investigations or other studies that require a high yield of early passage cells. Bovine thoracic aortas were obtained at the local slaughterhouse. The caudal extremity of the thoracic duct was identified in the periadventitial fat on the posterolateral side of the aortas caudal end, and injected with 0.1% Evans blue in saline (Fig. 1). As the thoracic duct, thus stained, was freed from most of the periadventitial fat, numerous small lymph nodes appeared. The lymph nodes never interrupted the course of the vessel, being connected with its lumen via short, thin collectors. The thoracic duct had no collateral branches; however, either of its extremities or the entire duct were sometimes duplicated. Both the extremities of the duct were cannulated with intravenous eannulae and the caudal extremity was dearly marked so that fluids could be infused in the normal direction of the lymph. The presence of valves prevented retrograde flow. The vessel was washed with phosphate buffered saline, filled with warm medium M199 (GIBCO, Life Technologies Ltd., Paisley, Scotland, UK) and incubated for 15 min at 37 ° C in a beaker containing warm saline. The vessel was then filled with 0.2% collagenase (Boehringer Mannheim, GmbH, W. Germany, 0.215 U/ mg) and incubated for 15 min at 37 ° C. After collecting the collagenase solution from the duct, the vessel was massaged to favor the detachment of LEC and washed with 30 ml of M199. All the effluents were spun for 10 min at 630 Xg. The cell pellet was resuspended in complete tissue culture medium (4): M199 containing 20% fetal bovine serum (GIBCO), 100 /.tg/ml endothelial ceil growth supplement (Sigma, St. Louis, MO), 100 /.tg/ml heparin (Sigma), 50 U/ml penicillin and 50 #g/ml streptomycin (GIBCO). The cells were seeded at a density of 1.6 × 104 cells/cm 2 into gelatin-coated plates or round glass cover slips, depending on intended use, and incubated in a humidified atmosphere with 5% CO2. The medium was changed 48 h after seeding and every other

Prophylactic role of acetyl-l-carnitine on knee lesions and associated pain in a rat model of osteoarthritis

AIMS in the present study, our aim was to validate in vivo the prophylactic role of acetyl-l-carnitine (ALC) using an established knee osteoarthritis (OA) animal model which mimics the pathological changes of OA in humans, targeting cartilage and causing chondrocyte death. MAIN METHODS animal model was obtained by an intra-articular injection of monosodium iodoacetate (MIA) into rat femorotibial joint space. Pain was measured in animals submitted to MIA model by paw pressure and compression behavioral tests in the presence or absence of ALC. KEY FINDINGS morphological analysis of knee-joint from MIA and ALC co-treated rats showed that the total pathological score attributed to histological findings was dramatically lower in rats treated with MIA in the presence of ALC. OA chondrocyte overexpression of pathogenic collagenase matrix-metallopeptidase-13 (MMP13) could be decreased in knee-cartilage from MIA/ALC rats; whereas type II collagen (COL2) expression level could be partially increased to control value. ALC twice daily treatment was able to attenuate pain in OA rat knee as revealed by mechanical behavioral tests. SIGNIFICANCE in our experiments, pain that is usually associated with OA, was correlated with the severity of histopathological findings. Our findings show that there is a place for ALC as chondroprotective agents in cartilage degradation and strongly support the prophylactic and therapeutic potentials of ALC in knee-OA patients.

Treatment of experimental esophagogastric myotomy with bone marrow mesenchymal stem cells in a rat model

Over the last 15 years, many studies demonstrated the myogenic regenerative potential of bone marrow mesenchymal stem cells (BM‐MSC), making them an attractive tool for the regeneration of damaged tissues. In this study, we have developed an animal model of esophagogastric myotomy (MY) aimed at determining the role of autologous MSC in the regeneration of the lower esophageal sphincter (LES) after surgery.

Characterization of lymphatic vessels in human peripheral neuropathies

Immunohistological studies on lymphatics’ topography in human peripheral nerve are few and recent. By D2-40 immunohistochemistry, we previously described lymphatics in epineurium of human sural nerve. Lymphangiogenesis is described in inflammation. In angiopathic and vasculitic neuropathies proliferation of epineurial blood capillaries is reported. The aim of our study is therefore to investigate the topography and the density of lymphatics in human peripheral nerve and to search possible correlation with blood capillary neovascularization in different neuropathies. We examined biopsied sural nerves of patients suffering from CIDP (chronic inflammatory demyelinating polyneuropathy), vasculitic neuropathy and non inflammatory axonal neuropathy. Immunohistochemistry for detection of lymphatic marker podoplanin (D2-40 antibody) and for general endothelial marker CD31, as well as for Schwann cells protein S-100, was carried out on serial cryostat sections. Morphometric analysis was performed. Lymphatic capillaries were detected in epineurium, most consistently in adjacency of main blood vessels. Occasionally lymphatics were dilated and repleted with mononuclear cells. Podoplanin was also expressed by perineurium and by Schwann cells. No lymphatics were observed endoneurially. Lymphatics showed a far lesser density than blood capillaries and increase of epineurial vascularization resulted significantly associated with higher density of lymphatics. Density variations of epineurial lymphatics, accompanying blood capillaries proliferation, suggest that lymphangiogenesis may occur in neuropathies, in response to inflammation/ischaemia. Lymphatics’ responsiveness to molecular microenvironment is indicated by their expression of growth factor receptors, such as VEGFR3. Lack of lymphatics in closed endoneurial environment is in agreement with analogous findings in brain. Non lymphatic expression of podoplanin is reported in several cell types of nervous system: normal and tumoral ependymal cells, perineurium and Schwann cells. As biological functions of podoplanin, involved in cell migration and cytoskeletal reorganization, are uncompletely understood, its localization on non lymphatic structures of peripheral nerve needs to be defined.

Sternal foramina :anatomy and clinical significance

The presence of one or more sternal holes is a congenital developmental anomaly that needs to be recognized and diagnosed to prevent accidental puncture of vital organs during procedures such as sternum biopsy or acupuncture. We present two cases of this bone anomaly characterized by the presence of sternal foramina that were found during tutorials for medical students in the anatomical museum” Leonetto Comparini “of University of Siena . Measurements of the foramina were carried out using a digital caliper and was subsequently made a photographic documentation.The first case shows a sternum with multiple oval foramina: one at level of body sterni with the larger diameter of mm 4,78 and two at xiphoid process with larger diameter of 8,83 and 7,44 mm respectively.The second case is a sternum with a single oval foramen at level of the lower part of body with a diameter of 12,8mm. In the fetus sternum cartilage is formed by two bars which merge with each other towards the eighth week of gestation forming the manubrium and the body of the sternum(1).At the tenth week of gestation .the subsequent ossification of the sternum takes place from six centers of ossification. The last part of the sternum to ossify in adulthood is the xyphoid process . A partial defect in the melting of cartilage bars can cause holes to form in the sternum. The incidence is between 3.1 to 27.4% in dried sterna (2). Sternal holes are observed in the manubrium, body, and in the xiphoid process, also if a highest incidence is verifyed in the xiphoid process. The presence of sternal holes may cause during sternal puncture the accidental punture of organs retrosternal as the heart and lungs with possible tamponade or pneumothorax .Moreover knowledge of this anomaly may be important in forensic medicine. The presence of a sternum holes may be mistakenly interpreted as penetrating traumatic injuries or bullet penetration. In conclusion, the recognition of this not uncommon anatomical abnormality is important for radiologists and in clinical and forensic medicine.

Muscle pathology patterns in possibly adjuvant related autoimmune/inflammatory syndrome (ASIA)

Growing evidence shows a link for biologically inert molecules, such as vaccine adjuvants and silicone implants, with the occurrence of autoimmunity-related disorders, defined as autoimmune/inflammatory syndrome induced by adjuvant-ASIA (1). Clinical conditions encompass siliconosis, the Gulf war syndrome, the macrophagic myofasciitis syndrome (MMF), post-vaccination phenomena and the spectrum of related syndromes is expanding (2). Involvement of skeletal muscle in ASIA is acknowledged in MMF, defined by long-term persistence of vaccine alum adjuvants within macrophages at sites of previous immunization. A few reports describe vaccine and silicone implants related autoimmune inflammatory myopathies (3). We carried out an immunopathological analysis of skeletal muscle biopsy in a case of MMF and two cases of possible ASIA myositis, chronologically subsequent to breast silicone implant. MMF showed the typical fascial/ perimysial macrophagic invasion, with no endomysial mononuclear infiltrates and fibral neolocalization of MHC-I complex restricted to the adjacency of macrophage deposits. The first myositis case presented with a subacute onset twenty years after an uneventful additive breast silicone implant. Endomysial inflammation, microangiopathy and multifocal fibral localization of MHC-II complex were observed. In the second patient, the onset of proximal weakness, myalgiae and a tenfold increase of creatinkinase levels occurred seven years after an unsuccessful additive mastoplasty, with rupture of prostheses and re-implantation three years later. Muscle biopsy, besides inflammation changes, showed peculiar myofibrillar disruption, with MHC-I reactive sarcoplasmic inclusions expressing several structural muscle proteins. Molecular pathogenesis of ASIA is yet undefined: genetical susceptibility is currently investigated (1,2). Due to the role of vaccines in medicine and the wide use of silicon medical devices, identification of their cause/effect link with autoimmunity is of great interest.

Immunohistochemical detection of myosin heavy chain isoforms in human cremaster muscle

Cremaster muscle (CM) forms a thin network of fascicles, around the spermatic cord and testis, connected by loose areolar tissue forming the cremasteric fascia. CM has a non somitic embryologic origin, as it derives from mesenchymal differentiation of the gubernacular tip (1).Thus it is not to be considered a passive extension of internal oblique muscle. CM is composed both of striated and smooth muscle cells; it is innervated by genitofemoral nerve (2). Its striated fibres, in contrast with skeletal muscles, present with a multifocal innervation by multiple neuromuscular synapses (3). Myosin isoforms are the major determinant of the contractile and biochemical heterogeneity of skeletal muscle fibers. Non somitic muscles, such as extrinsic ocular muscles, show a distinct pattern of myosin heavy chains distribution. The aim of our study was to characterize the expression of myosin isoforms in CM fascicles; biopsy samples were obtained from cases of cryptorchidism, retractile testis and inguinal hernia, undergoing surgery. Immunohistochemistry confirmed the previously identified type 1 predominance (1) and showed a high occurrence of hybrid fibres, coexpressing two or more myosin isoforms. In contrast with age-matched limb muscles, persistence of developmental/neonatal myosin heavy chains was detected, beyond the determined timecourse of physiological shifting from immature isoforms (4). On the basis of shared peculiar embryological derivation, expression of superfast extraocular myosin MyH13 was also investigated on CM specimens, showing sarcoplasmic reactivity, undetectable in limb muscles. The high share of hybrid fibres, the persistence of immature myosin and MyH13/MyHCslow coexpression are peculiar features, suggesting a functional/biochemical individuality of CM, related with multiple innervation and distinct embryological development.

Treatment of lower oesophageal sphincter damage with bone marrow derived mesenchymal stem cells

Background. The incompetence of lower esophageal sphincter (LES) with consequent exposition of the esophageal mucosa to gastric acid leads to gastroesophageal reflux disease. The aim of this study was to evaluate whether intralesional injections of Bone Marrow Mesenchymal Stem Cells (BM-MSC), which proved useful in the treatment of urinary (1) and anal (2) incontinence, could also help muscle regeneration at the site of surgical myotomy of rat LES. Methods. 24 inbred Wistar Furth rats were divided into three groups: group A underwent a sham operation followed by saline injection; group B LES myotomy plus saline injection; group C LES myotomy followed by an intra-sphincteric injection of culture-expanded syngeneic BM-MSC. At day 30, histological and morphometric analysis were performed on metacrylate embedded samples. GFP positive MSC isolated from transgenic rats were moreover used to track the cells in the injured area in cryostat sections at days 7, 14 and 30 after the lesion. Cryostat sections were also used for immunohistochemical detection of striated and smooth muscular markers (a actinin, a smooth muscle actin, calponin). Results. At day 30 of surgery, the muscle area fraction (MAF) at the site of LES myotomy was significantly higher in group C compared to group B animals (p<0.05) and contained a high number of small irregularly arranged smooth muscle cells, sometimes grouped in clusters. GFP positive cells could be tracked at the periphery of the lesion at day 7 and also inside the lesion at day 14 of surgery when the damaged area, as evidenced by specific antibodies, was still devoid of smooth or striated muscle cells. At day 30, the lesion was recognizable only as a disorganized area at the periphery of the muscular layer. At this time clusters of GFP positive cells, unstained by muscular specific antibodies, could still be detected at the periphery and sometimes also in the center of the muscular layer. Conclusions. BM-derived MSC, improving muscle regeneration of surgically myotomy LES in the rat, may represent a valuable tool in the treatment of LES injury. 1) Y.Kinebuchi et al. Int.J.Urol. 2010, 17:359-68 2) B.Lorenzi et al.Dis Colon Rectum 2008, 51:411-20.

A Novel Monoclonal Antibody Specific for Lymphatic Endothelium

The difficulty of identifying and differentiating lymphatic and blood microvessels in tissue sections can be overcome by a monoclonal antibody specific for lymphatic endothelium. Unfortunately, the only known antibody also reacts with the endothelium of some blood vessels. The technique of double immunization (passive, with an antiserum to blood endothelium, and active, with a suspension of lymphatic endothelial cells) was, therefore, used to increase the chances of recognizing specific lymphatic antigens by the mouse immune system. The monoclonal antibody obtained, LyMAb, a G1 immunoglobulin, reacted strongly with the endothelium of bovine thoracic duct, mesenteric collecting vessels and lymphatic vessels of gall-bladder and lymph nodes and moderately with those of the intestinal wall. Blood vessels (intercostal arteries, azygos vein and blood microvessels of all organs tested) were consistently negative. The antibody was species-specific and did not react with formalin-fixed, paraffin-embedded sections. Cross-reactivity was limited to some connective tissue fibres and scattered cells in the lymph node parenchyma, intestinal villi and hepatic lobules.