Mauro Pluderi

Angiogenesis and Invasion in Gliomas

Angiogenesis and tumor cell invasion are pathophysiological processes playing a pivotal role in glioma development and growth since the earliest phase. Angiogenesis and tumor invasion both can be considered as an invasive process in which cells are activated, and move away from their initial location, by modyfing the adhesiveness with the extracellular matrix, expressing new adhesion molecules, and degrading the extracellular matrix components by the active secretion of proteases. This process requires a complex cross-talking between endothelial and tumor cells, extracellular matrix components, and cellular elements of the host microenviroment. Both processes are under the tight regulation of a balance between stimulating and inhibiting factors. The existence of common mechanisms of regulation and the presence of naturally occurring factors that inihibit angiogenesis and invasion, makes the inhibition of both processes possible. Tumor cells may develop adapting mechanims that can allow the tumor to partially escape to the treatment, particularly when only one mechanism or one process is inhibited. The ideal treatment should simultaneously affect both angiogenesis and invasion, by the isolation or development of novel therapeutics capable of influencing both processes. As their efficacy seems also be dependent on the mode of delivery, additional studies are also needed to improve these modalities, in order to ultimately improve the extent and the duration of the therapeutic response. The most widely used in vitro and in vivo models to study angiogenesis and invasion are also discussed.

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin‐derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP‐labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L‐lactide and trimethylene carbonate (PLA‐TMC) copolymer and a collagen‐based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S‐100 and glial fibrillary acidic protein. RT‐PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.

Combinatorial administration of molecules that simultaneously inhibit angiogenesis and invasion leads to increased therapeutic efficacy in mouse models of malignant glioma.

Purpose: We investigated the ability of the combinatorial administration of different inhibitors with activities on glioma angiogenesis, migration, and proliferation to produce a prolonged inhibition of glioma growth. Experimental Design: We combined inhibitors affecting solely tumor angiogenesis (PF-4/CTF, cyclo-VEGI) or inhibitors affecting both angiogenesis and invasion together (PEX, PF-4/DLR). Results: When administered in combination, these drugs produced a prolonged and increased inhibition of glioma growth independently from the type of inhibitor used. The combinatory administration was more effective than the administration of a single inhibitor alone, and a strong therapeutic response was reached with a significantly lower amount of protein. The strongest inhibition was observed when human PEX and PF-4/DLR, which affect both glioma angiogenesis and invasion by separate mechanisms, were combined. Conclusions: This supports the concept that prolonged glioma growth inhibition can be achieved by simultaneous delivery of molecules that target both tumor and endothelial cells and acting by separate mechanisms.

Relationship between systemic glucose and cerebral glucose is preserved in patients with severe traumatic brain injury, but glucose delivery to the brain may become limited when oxidative metabolism is impaired: implications for glycemic control.

Objective:To clarify the dynamics of glucose delivery to the brain and the effects of changes in blood glucose after severe traumatic brain injury. Design:Retrospective analysis of a prospective observational cohort study. Setting:Neurosurgical intensive care unit of a university hospital. Patients:Seventeen patients with acute traumatic brain injury monitored with cerebral and subcutaneous microdialysis. Interventions:None. Measurements and Main Results:For continuous, accurate systemic monitoring, glucose was measured in the interstitial space of subcutaneous adipose tissue using microdialysis, and 39 specific episodes of spontaneous rises in glucose were identified. During these episodes, there was a significant positive linear relationship between systemic glucose levels and brain glucose concentrations measured by microdialysis (p < .0001). The basal lactate/pyruvate ratio, with a threshold of 25, was adopted to distinguish between disturbed and presumably preserved cerebral oxidative metabolism. Using normal vs. elevated lactate/pyruvate ratio as variable factor, the relationship between brain and systemic glucose during the episodes could be described by two significantly distinct parallel lines (p = .0001), which indicates a strong additive effect of subcutaneous glucose and lactate/pyruvate ratio in determining brain glucose. The line describing the relationship under disturbed metabolic conditions was lower than in presumably intact metabolic conditions, with a significant difference of 0.648 ± 0.192 mM (p = .002). This let us to accurately predict that in this situation systemic glucose concentrations in the lower range of normality would result in critical brain glucose levels. Conclusions:The linear relationship between systemic and brain glucose in healthy subjects is preserved in traumatic brain-injured patients. As a consequence, in brain tissue where oxidative metabolism is disturbed, brain glucose concentrations might possibly drop below the critical threshold of 0.8 mM to 1.0 mM when there is a reduction in systemic glucose toward the lower limits of the “normal” range.

IS20I, a specific αvβ3 integrin inhibitor, reduces glioma growth in vivo

OBJECTIVE The biological features of malignant gliomas include high cell proliferation, extensive local infiltration of tumor cells into normal brain, and marked neovascularization. alphavbeta3 integrin is highly expressed in malignant gliomas and plays a role in glioma growth. This article investigates the in vitro and in vivo effects of a synthetic alphavbeta3 integrin inhibitor called IS20I on human malignant gliomas. METHODS The in vitro effects of IS20I were studied by performing adhesion assays, competition studies, semi-in vivo angiogenic assays, and migration and proliferation assays. For the in vivo experiments, IS20I was administered systemically in nude mouse intracranial and subcutaneous malignant glioma models. RESULTS IS20I reacted selectively to alphavbeta3 integrin in glioma cells and tissues. In vitro, IS20I strongly inhibited angiogenesis and simultaneously exhibited potent antimitotic and antimigratory effects on numerous tumor and endothelial cell lines. In addition, at high concentrations, IS20I induced endothelial and tumor cell apoptosis. In vivo, when IS20I was administered intraperitoneally in subcutaneous and intracranial nude mouse glioma models, it potently reduced malignant glioma growth. Inhibition levels of 76 and 82% were observed at concentrations of 1 and 5 mg/kg, respectively, in the U87 intracranial model. The suppression of tumor growth is associated with a decrease in tumor vascularity, an increase in apoptosis, and a decrease in tumor cell proliferation. CONCLUSION This work expands the understanding of the effects of anti-alphavbeta3 integrin inhibitors on malignant gliomas. In addition to direct proapoptotic and antiangiogenic effects, IS20I inhibits tumor and endothelial cell proliferation and migration, resulting in a potent inhibition of glioma growth in vivo.

Mitochondrial changes in platelets are not related to those in skeletal muscle during human septic shock

Platelets can serve as general markers of mitochondrial (dys)function during several human diseases. Whether this holds true even during sepsis is unknown. Using spectrophotometry, we measured mitochondrial respiratory chain biochemistry in platelets and triceps brachii muscle of thirty patients with septic shock (within 24 hours from admission to Intensive Care) and ten surgical controls (during surgery). Results were expressed relative to citrate synthase (CS) activity, a marker of mitochondrial density. Patients with septic shock had lower nicotinamide adenine dinucleotide dehydrogenase (NADH)/CS (p = 0.015), complex I/CS (p = 0.018), complex I and III/CS (p<0.001) and complex IV/CS (p = 0.012) activities in platelets but higher complex I/CS activity (p = 0.021) in triceps brachii muscle than controls. Overall, NADH/CS (r2 = 0.00; p = 0.683) complex I/CS (r2 = 0.05; p = 0.173), complex I and III/CS (r2 = 0.01; p = 0.485), succinate dehydrogenase (SDH)/CS (r2 = 0.00; p = 0.884), complex II and III/CS (r2 = 0.00; p = 0.927) and complex IV/CS (r2 = 0.00; p = 0.906) activities in platelets were not associated with those in triceps brachii muscle. In conclusion, several respiratory chain enzymes were variably inhibited in platelets, but not in triceps brachii muscle, of patients with septic shock. Sepsis-induced mitochondrial changes in platelets do not reflect those in other organs.

Inhibition of FGF receptor activity in glioma implanted into the mouse brain using the tetracyclin-regulated expression system.

We have investigated growth and vascularization of malignant glioma in mice upon conditional inhibition of fibroblast growth factor (FGF) receptor activity. C6 rat glioma cells were transfected with a dominant-negative fibroblast growth factor receptor-2 (FGFR2-DN) cDNA under the control of a tetracycline-regulated expression promoter (tet off) and implanted in the brain of immunodeficient mice. Magnetic resonance imaging analysis showed a significant decrease in tumor growth 14 days after implantation when FGFR2-DN was expressed compared to control. This size difference disappeared after 20 days. However, after 20 days, tumor and endothelial cells apoptosis were higher in the FGFR2-DN group and consequently angiogenesis was decreased whereas tumor cells were similarly associated with blood vessels at the tumor periphery. Pericyte coverage was not different between the two groups but a higher amount of pericytes not associated with vessels was found in the FGFR2-DN expressing group. This demonstrates, that conditional expression of inhibitor of FGF receptor activity in gliomas implanted in the brain of immunodeficient mice can be achieved efficiently, and that FGFs are major players in glioma development and in glioma angiogenesis.

Impact of cerebrovascular disease on the surgical treatment of idiopathic normal pressure hydrocephalus

OBJECTIVE:The influence of cerebrovascular disease (CVD) on the short- and long-term results of surgery was evaluated in a series of consecutive patients with idiopathic normal-pressure hydrocephalus (iNPH). METHODS:Patients with suspected iNPH admitted to our department between June 1996 and June 2003 were evaluated with four clinical and handicap scales. CVD and risk factors for vascular disease were rated. All patients underwent intracranial pressure monitoring via a spinal catheter. Sixty-six patients received a ventriculoperitoneal shunt with a programmable valve. Prospective assessments were programmed at 2 weeks and 3 months after surgery (short-term follow-up). Long-term follow-up evaluations were arranged in June 2004 with patients and/or relatives and health/home care assistants. RESULTS:At the short-term follow-up examination, a significant clinical improvement was globally present in 89% of the patients (P < 0.05). CVD, such as leucoaraiosis or previous strokes, were present in 71% of the patients. Patients both with and without CVD and/or risk factors for vascular disease presented a significant improvement (P < 0.05) after shunting; 85 and 100% of the patients with and without CVD, respectively. At the long-term follow-up examination (mean, 52 ± 24.8 mo), 24% of the patients were dead and 8% had experienced stroke. Globally, 60% of the patients were still improved (P < 0.05); 52 and 79% of the patients with and without CVD, respectively. CONCLUSION:A high success rate in treatment of iNPH is possible in patients with and without CVD. Despite poorer short- and long-term treatment outcome of iNPH patients with CVD, a long-lasting improvement in their quality of life favors surgery.

Stem cell salvage of injured peripheral nerve

We previously developed a collagen tube filled with autologous skin-derived stem cells (SDSCs) for bridging long rat sciatic nerve gaps. Here we present a case report describing a compassionate use of this graft for repairing the polyinjured motor and sensory nerves of the upper arms of a patient. Preclinical assessment was performed with collagen/SDSC implantation in rats after sectioning the sciatic nerve. For the patient, during the 3-year follow-up period, functional recovery of injured median and ulnar nerves was assessed by pinch gauge test and static two-point discrimination and touch test with monofilaments, along with electophysiological and MRI examinations. Preclinical experiments in rats revealed rescue of sciatic nerve and no side effects of patient-derived SDSC transplantation (30 and 180 days of treatment). In the patient treatment, motor and sensory functions of the median nerve demonstrated ongoing recovery postimplantation during the follow-up period. The results indicate that the collagen/SDSC artificial nerve graft could be used for surgical repair of larger defects in major lesions of peripheral nerves, increasing patient quality of life by saving the upper arms from amputation.

Third Ventriculostomy in Late-onset Idiopathic Aqueductal Stenosis Treatment: A Focus on Clinical Presentation and Radiological Diagnosis

Endoscopic third ventriculostomy (ETV) is considered the gold standard treatment for obstructive hydrocephalus due to partial or complete obstruction of cerebrospinal fluid (CSF) ventricular pathways caused by mass lesions. However long-term efficacy of this procedure remains controversial as treatment of chronic adult hydrocephalus due to stenosis of Sylvian acqueduct [late-onset idiopathic aqueductal stenosis (LIAS)]. The authors describe clinical presentation, diagnostic investigations in patients affected by LIAS, and define their clinical and radiological outcome after ETV. From January 2003 to December 2008, 13 consecutive LIAS patients treated by ETV were retrospectively reviewed. Pre- and post-operative clinical and radiological findings, including conventional and phase-contrast (PC) cine magnetic resonance imaging (MRI) were investigated. ETV was successfully performed in all patients. Patients neurological condition improved. No one required a second ETV procedure or shunt implantation. Clinical and radiological results reveal a satisfactory outcome of LIAS patients treated by ETV. At follow-up a clinical improvement could be demonstrated in all cases. Selection criteria of LIAS patients seem to be crucial to obtain satisfactory and long-lasting results. Even in elderly patients with chronic hydrocephalus, ETV can be considered the treatment of choice.