On the Connection Between Geometry and Statically Determined Membrane Stresses in the Human Cornea.

Under the action of the intraocular pressure (IOP), the human cornea is stressed and deforms acquiring a quasi-spherical configuration. If the stressed configuration is known, and the cornea is regarded as a membrane, disregarding flexural behaviors with an equilibrium analysis only is possible to estimate the distribution of the average stress across the thickness. In the cornea, the action of the intraocular pressure is supported by collagen fibrils, immersed into an elastin-proteoglycan matrix, and organized in a very precise architecture to provide the necessary confinement and transparency to the light. With the goal of understanding the static consequences of shape modifications due to pathological dilatation (ectasia), we present a simplified stress analysis of the human cornea modeled as a membrane. A numerical investigation over 40 patient-specific corneas (20 normal and 20 ectatic) is carried out to establish a relationship between the physiological geometry and the distribution of the membrane stresses, and to assess the possibility to obtain information on the stress state based on topographic images only. Comparative analyses reveal that, with respect to normal corneas, in ectatic corneas the pattern of the principal stress lines is modified markedly showing a deviation from the hypothetical dominant orientation of the collagen fibrils. The rotation of the principal stress with respect to the fibril orientation can be thought as responsible of the transmission of a large amount of shear stresses onto the elastin-proteoglycan matrix. The anomalous loading of the matrix could be correlated to the evolution of time-dependent shape modifications leading to ectasia.

A microstructural model of cross-link interaction between collagen fibrils in the human cornea.

We propose a simplified micromechanical model of the fibrous reinforcement of the corneal tissue. We restrict our consideration to the structural function of the collagen fibrils located in the stroma and disregard the other all-important components of the cornea. The reinforcing structure is modelled with two sets of parallel fibrils, connected by transversal bonds within the single fibril family (inter-cross-link) and across the two families (intra-cross-link). The particular design chosen for this ideal structure relies on the fact that its ability to sustain loads is dependent on the degree of the cross-link and, therefore, on the density and stiffness of the bonds. We analyse the mechanical response of the system according to the type of interlacing and on the stiffness of fibres and bonds. Results show that the weakening of transversal bonds is associated with a marked increase of the deformability of the system. In particular, the deterioration of transversal bonds due to mechanical, chemical or enzymatic reasons can justify the loss of stiffness of the stromal tissue resulting in localized thinning and bulging typically observed in keratoconus corneas. This article is part of the theme issue 'Rivlin's legacy in continuum mechanics and applied mathematics'.

Centella asiatica and lipoic acid, or a combination thereof, inhibit monocyte adhesion to endothelial cells from umbilical cords of gestational diabetic women.

BACKGROUND AND AIMS: Diabetes mellitus is associated with inflammatory endothelial activation and increased vascular leukocyte adhesion molecule expression, both playing a prominent role in the development of vascular complications. Centella asiatica (CA) and Lipoic Acid (LA) have shown anti-inflammatory and anti-oxidant properties in a variety of experimental models; however, their action on human umbilical vein endothelial cells (HUVECs), chronically exposed to hyperglycemia and pro-inflammatory environment during pregnancy, is still unknown. METHODS AND RESULTS: In HUVECs from umbilical cords of gestational diabetic (GD) or healthy (C) women, both CA and LA affected tumor necrosis factor-α (TNF-α)-induced inflammation, being associated with a significant decrease in vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression (western blot) and exposure (flow cytometry), as well as monocyte-HUVECs interaction (adhesion assay). Notably, this was associated with a significant reduction of an index of nitro-oxidative stress, such as the intracellular peroxynitrite levels (fluorescence detection by cytometric analysis), Mitogen-Activated Protein kinase (p44/42 MAPK) expression/phosphorylation levels and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) cytoplasm-nucleus translocation (flow cytometry). Overall our results indicate that both CA and LA used separately, and even better when combined, are effective to reduce the inflammatory response in TNF-α-treated HUVECs. Notably, this was more significant in GD than in C-HUVECs and also evident at baseline. CONCLUSION: In conclusion, our in vitro study demonstrates that both CA and LA, or a combination thereof, are able to mitigate the potentially dangerous effects on the endothelium of chronic exposure to hyperglycemia in vivo.

Transcriptome analysis of human primary endothelial cells (HUVEC) from umbilical cords of gestational diabetic mothers reveals candidate sites for an epigenetic modulation of specific gene expression.

Within the complex pathological picture associated to diabetes, high glucose (HG) has "per se" effects on cells and tissues that involve epigenetic reprogramming of gene expression. In fetal tissues, epigenetic changes occur genome-wide and are believed to induce specific long term effects. Human umbilical vein endothelial cells (HUVEC) obtained at delivery from gestational diabetic women were used to study the transcriptomic effects of chronic hyperglycemia in fetal vascular cells using Affymetrix microarrays. In spite of the small number of samples analyzed (n=6), genes related to insulin sensing and extracellular matrix reorganization were found significantly affected by HG. Quantitative PCR analysis of gene promoters identified a significant differential DNA methylation in TGFB2. Use of Ea.hy926 endothelial cells confirms data on HUVEC. Our study corroborates recent evidences suggesting that epigenetic reprogramming of gene expression occurs with persistent HG and provides a background for future investigations addressing genomic consequences of chronic HG.

Features of endothelial dysfunction in umbilical cord vessels of women with gestational diabetes.

BACKGROUND AND AIMS: Gestational diabetes (GDM) is associated with increased oxidative stress and overexpression of inflammatory cytokines, both of which might lead to endothelial dysfunction and vascular disease. As such, GDM could be viewed as a sort of 'short lived' metabolic syndrome. As umbilical cord vessels represent a suitable model for the study of vascular alterations brought about by GDM, the aim of the present work was to characterize the phenotype of human umbilical vein endothelial cells (HUVECs) chronically exposed to hyperglycaemia and to a pro-inflammatory environment during pregnancy so as to identify molecular modifications of cellular homoeostasis eventually impacting on endothelial dysfunction. METHODS AND RESULT: Tissue specimens and HUVECs were obtained from umbilical cords of GDMand control women. As compared to controls, GD-HUVEC exhibited enhanced monocyte adhesion and vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1(ICAM-1) expression and exposure on plasma membrane after tumour necrosis factor-alpha(TNF-α) stimulation (Western blot, flow cytometer). As compared to control cells, GD-HUVEC in basal conditions exhibited enhanced monocyte adhesion, nitric oxide synthase (NOS) expression and activity (eNOS Real-Time polymerase chain reaction, Western Blot for eNOS total protein and monomers/dimers ratio, conversion of [3H]-L-arginine in [3H]-L-citrulline), increased O(-)(2)egeneration together with increased NT levels (immunofluorescence) and reduced NO bioavailability(guanosine 3',5'-monophosphate (cGMP) production, EIA). Furthermore, immunohistochemistry revealed increased eNOS and NT immunoreactivity in GD umbilical cords. CONCLUSION: Endothelial cells exposed in vivo even transiently to hyperglycaemia, oxidative stress and inflammation exhibit durable pro-atherogenic modifications.

A discrete-to-continuum model for the human cornea with application to keratoconus.

We introduce a discrete mathematical model for the mechanical behaviour of a planar slice of human corneal tissue, in equilibrium under the action of physiological intraocular pressure (IOP). The model considers a regular (two-dimensional) network of structural elements mimicking a discrete number of parallel collagen lamellae connected by proteoglycan-based chemical bonds (crosslinks). Since the thickness of each collagen lamella is small compared to the overall corneal thickness, we upscale the discrete force balance into a continuum system of partial differential equations and deduce the corresponding macroscopic stress tensor and strain energy function for the micro-structured corneal tissue. We demonstrate that, for physiological values of the IOP, the predictions of the discrete model converge to those of the continuum model. We use the continuum model to simulate the progression of the degenerative disease known as keratoconus, characterized by a localized bulging of the corneal shell. We assign a spatial distribution of damage (i.e. reduction of the stiffness) to the mechanical properties of the structural elements and predict the resulting macroscopic shape of the cornea, showing that a large reduction in the element stiffness results in substantial corneal thinning and a significant increase in the curvature of both the anterior and posterior surfaces.

Mechanical characterization of porcine corneas.

An experimental program has been carried out in order to investigate the mechanical behavior of porcine corneas. We report the results of inflation tests on the whole cornea and uniaxial tests on excised corneal strips, performed on 51 fresh porcine eyes. Uniaxial tests have been performed on specimens cut from previously inflated corneas. The cornea behavior is characterized by means of elastic stiffness, measured on both average pressure-apex displacement and average uniaxial stress-strain curves; and by means of transversal contraction coefficient, peak stress, and failure stress measured on uniaxial stress-strain curves. Uniaxial tests performed on excised strips allowed to measure the anisotropy in the corneal stiffness and to compare the stiffness of the cornea with the one of the sclera. Viscous properties of the cornea have been obtained through uniaxial relaxation curves on excised corneal strips. The relevant geometrical parameters have been measured and, with the aid of the elastic thin shell theory, a stress-strain curve has been derived from the average inflation test data and compared with similar data available in the literature. The experimental system has been developed in view of future applications to the mechanical testing of both porcine and human corneas.

Numerical estimation of stress and refractive power maps in healthy and keratoconus eyes.

Keratoconus is an eye condition caused by localized thinning of the corneal tissue, which leads to a characteristic cone-shaped protrusion of the cornea. We investigate the mechanical behavior of keratoconus and suspect keratoconus corneas versus healthy corneas by using patient-specific finite element models. Patient-specific geometries of the corneas are obtained from diagnostic images provided by corneal topographer, transformed into solid models, and discretized in hexahedral elements. For the diseased corneas, a suitable reduction of the stiffness is applied within a limited region of the cornea around the conus. After the identification of the stress-free configuration, the models are used to simulate pressurization tests up to 40 mmHg. The material parameters have been estimated within the stress-free configuration identification procedure. As expected, numerical results reveal a more compliant behavior for the diseased corneas in terms of apex displacement plots as a function of the intraocular pressure, with diseased corneas experiencing up to 44% increase in apex displacement compared to healthy corneas. The maps of the stress confirm, for the diseased corneas, a marked increase of the maximum tensile stress, on both anterior and posterior surfaces, to be ascribed mainly to the reduction of the corneal thickness. Stress maps also show, for keratoconus corneas, a marked increase of the ratio between posterior and anterior tensile stress in the conus. Numerical analyses are used to construct the refractive power maps, revealing clearly that the maximum dioptric power in keratoconus corneas is at the center of the cone-shape rather than at the apex.

Numerical modelling of fracture in human arteries.

We present 3D finite element models of atherosclerotic arteries, used to investigate the influence of the geometry and tissue properties on the plaque rupture caused by overexpansion. We adopted a geometry reconstructed from a contiguous set of in vitro magnetic resonance images of a damaged artery. The artery wall is divided in three layers (adventitia, media and intima) and is discretized into tetrahedral finite elements. The artery material is described with a hyperelastic two-fiber anisotropic model proposed by Holzapfel et al. 2000. A new constitutive framework for arterial wall mechanics and a comparative study of material models. J Elasticity 61(1):1-48, while the plaque is assumed to be transversely isotropic. Cracks induced by mechanical actions are represented through cohesive surfaces, and are allowed to develop along solid elements boundaries only. Fractures are explicitly introduced in the discretized model at the locations where the tensile strength of the material is reached.

[Reduced nitric oxide bioavailability in chronic renal failure: a new factor of progression?]. / Ridotta biodisponibilità di ossido nitrico nell'insufficienza renale cronica: un nuovo fattore di progressione?

Nitric oxide (NO) is a gaseous free radical and an important molecular mediator of many physiologic processes in virtually every organ. NO is produced from L-arginine by nitric oxide synthase (NOS). This enzyme is expressed as 3 isoforms, all of which have been isolated from the kidney: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). At present it is very difficult to measure authentic nitric oxide in vivo; a way to circumvent the difficulties is to study the effects of NOS stimulation and subsequent nitric oxide release directly by measurement of the resulting changes in vascular tone. In the kidney and vasculature, NO plays fundamental roles in the control of systemic and intrarenal hemodynamics, the tubuloglomerular feedback response, pressure natriuresis, release of sympathetic neurotransmitters and renin, and tubular solute and water transport. Chronic renal failure (CRF) is a state of NO deficiency secondary to decreased NO production and/or increased bioinactivation of NO by reactive oxygen species. The purpose of this review is to examine the functions of NO in the kidney, and to discuss the effects of NO deficiency in the progression of chronic kidney disease.

A modified approach to horizontal augmention of soft tissue around the implant: omega roll envelope flap. Description of surgical technique.

OBJECTIVE: The flap's design, as applied to implant surgery procedures for implant-prosthetic therapy, takes inspiration from the roll technique of Abrams (1980) and subsequent modifications. This article describes a modified flap design for the correction of horizontal alveolar mucosa defects in implant-supported rehabilitation with one-stage and two-stage approache. The omega roll envelope flap (OREF) is a roll flap combined with a modified pouch technique. The goal of this type of flap design is to correct localized horizontal alveolar ridge defects and augment peri-implant soft tissue thickness. DESIGN: OREF is a flap technique that avoids harvesting autologous connective tissue from another donor site by using the supracrestal connective tissue of the implant surgical site. The proposed technique allows for increased horizontal buccal soft tissue thickness during implant-prosthetic rehabilitation. DISCUSSION: The OREF technique is practical for one- and two-stage implant approaches, and when it is applied with an immediate non-functional loading procedure, this technique can optimize surgical and prosthesis chair times. This technique has shown advantages as maximizes the amount of connective tissue that can be rolled within the buccal flap. The OREF technique can be applied with a one-technique can be applied with a one-stage or two-stage implant-prosthetic approach.

The prominent role of p38 mitogen-activated protein kinase in insulin-mediated enhancement of VCAM-1 expression in endothelial cells.

Insulin levels are a marker for cardiovascular events, but the link between hyperinsulinemia and atherosclerosis is poorly understood. We previously showed that insulin increases monocyte-endothelial interactions and the endothelial expression of the pro-atherogenic vascular cell adhesion molecule-1 (VCAM-1). The aim of this study is to examine molecular mechanisms involved in the effect of insulin on VCAM-1 expression. Human umbilical vein endothelial cells (HUVEC) were incubated with insulin (0-24 h)+/- inhibitors of signaling pathways potentially involved. At pathophysiological concentrations (10(-9)-10(-7) M), insulin selectively induced VCAM-1 expression. The p38 mitogen activated protein(MAP) kinase inhibitors SB203580 and SB202190, and partially the c-Jun NH2-terminal kinase (JNK) inhibitor SP600127, decreased insulin effect on VCAM-1. Gene silencing by small interfering RNA significantly reduced the expression of p38MAP kinase, and this was accompanied by suppression of insulin-stimulated VCAM-1 expression. Treatment with insulin also led to the activation of NF-KB and induction of IKB-alpha phosphorylation, thus accounting for NF-KB translocation into the nucleus. Co-treatment of HUVEC with insulin and SB202190 strongly reverted the stimulatory effect of insulin on NF-KB activation, thus establishing a link between NF-KB activation and p38MAPkinase-mediated induction of VCAM-1 by insulin. In conclusion, pathophysiological insulin concentrations increase VCAM-1 expression and activate NF-KB. This mostly occurs through stimulation of p38MAP kinase.

A model for the human cornea: constitutive formulation and numerical analysis.

Abstract The human cornea (the external lens of the eye) has the macroscopic structure of a thin shell, originated by the organization of collagen lamellae parallel to the middle surface of the shell. The lamellae, composed of bundles of collagen fibrils, are responsible for the experimentally observed anisotropy of the cornea. Anomalies in the fibril structure may explain the changes in the mechanical behavior of the tissue observed in pathologies such as keratoconus. We employ a fiber-matrix constitutive model and propose a numerical model for the human cornea that is able to account for its mechanical behavior in healthy conditions or in the presence of keratoconus under increasing values of the intraocular pressure. The ability of our model to reproduce the behavior of the human cornea opens a promising perspective for the numerical simulation of refractive surgery.

Plasminogen activator inhibitor type 1 is increased in the arterial wall of type II diabetic subjects.

Plasma plasminogen activator inhibitor type 1 (PAI-1) increases in diabetes, and this might contribute to decreased fibrinolysis and accelerated atherosclerosis. Increased PAI-1 levels in the vessel wall could decrease local fibrinolysis and elevate thrombus formation and the unfavorable evolution of atherosclerotic plaques. High glucose increases PAI-1 synthesis in arterial wall cells in culture, and aortic wall PAI-1 levels have been found to be elevated in diabetic animals. However, arterial wall PAI-1 levels have not been investigated in diabetic subjects. Therefore, the aim of this study was to determine the effect of diabetes on PAI-1 levels in the arterial wall. Blood samples and small tissue specimens from the mammary artery were obtained from 11 diabetic and 10 nondiabetic subjects who underwent coronary artery bypass graft surgery. PAI-1 antigen localization in the arterial wall was obtained by immunohistochemistry and was read by laser scanning confocal microscopy; plasma fibrinolytic activity was measured by lysis of fibrin plates; and PAI-1 activity was assessed by a chromogenic method. PAI-1-related immunofluorescence was increased in the arterial wall of diabetic patients, whereas plasma fibrinolysis was reduced. These data provide evidence that diabetes is associated with increased PAI-1 in the arterial wall. This might be an important factor for increased cardiovascular risk and unfavorable plaque evolution in diabetes.

Differences in the glutathione system of cultured aortic smooth muscle cells from young and aged rats.

We studied the relation between the glutathione (GSH) system and cell proliferation in a model of smooth muscle cells (SMC) derived from the thoracic aorta of 4-6-week-old (young) and 15-month-old (aged) rats. SMC from aged rats showed greater levels of total non-protein thiol compounds (T-SH), increased glutathione transferase (GST) and increased glutathione reductase (GSSG-Red) activities compared with cells from young rats. These changes were associated with an increased proliferation rate of SMC from aged rats. To evaluate the role of GSH on cell proliferation better, a specific inhibitor of gamma-glutamyl-cystein synthetase, DL-buthionine-SR-sulphoximine (BSO) was used. BSO showed a dose-dependent inhibition of cell growth, with an IC50 of 10(-4) M, after 48-72 h of incubation. Removal of BSO restored cell growth, further suggesting a link between GSH levels and vascular cell proliferation. The inhibitory effect of BSO was about two times greater on SMC from young than on SMC from aged rats. BSO showed 56% inhibition on the proliferation of SMC from young rats and 32% inhibition on SMC from aged rats (10(-4) M, 72 h of incubation). A parallel reduction of GSH levels of 38% and 19% for SMC from young and aged rats, respectively, was observed, suggesting that age-related factors may influence the involvement of GSH system in cell proliferation.

Costs of high-dose chemotherapy and peripheral blood progenitor cell autograft for breast cancer.

The aim of the study was to analyze the real cost of single or tandem high-dose chemotherapy (HDC) and peripheral blood progenitor cell autologous transplant (PBPCT) in patients with breast cancer. We analyzed the costs of 40 PBPCT performed in 20 patients. Tandem transplant was planned for each patient. Resources used and direct costs were identified for each patient. The study was carried out using the hospital perspective and monetary values were reported in 1999 Euro. The mean cost of whole procedure for single transplant was 20,816.63 Euro, while the mean cost of tandem transplant was 38,770.83 Euro. The cost distribution in the two groups was similar: the most expensive phase of procedure was the supportive phase post transplant (about 60% of total cost), with the categories of cost most represented being professional fees (about 28%) and pharmacy (about 35%). Awaiting more convincing trials of the clinical advantage of HDC in breast cancer, our analytical evaluation of transplant costs for different therapeutic options, single or tandem, permits identification of the most expensive categories in order to intervene for cost savings.

Docetaxel and epirubicin plus G-CSF as mobilizing treatment to support high-dose chemotherapy in breast cancer.

BACKGROUND: In order to combine an active regimen with a simultaneous efficient mobilization of peripheral blood precursor cells (PBPC), we explored the combination of Docetaxel 75 mg/m2 and Epirubicin 120 mg/m2 with G-CSF 5 mcg/Kg/day s.c. to mobilize PBPC in breast cancer patients to support high-dose chemotherapy (HDC). PATIENTS AND METHODS: Forty patients were enrolled: 27 high risk and 13 metastatic. The entire procedure, including chemotherapy and PBPC collection, was on an outpatient basis. RESULTS: The median day of starting apheresis was day +10 (range 10-12) and the average value of circulating CD34+ cells at peak was 175/microliter (range 33-403). The median yield of CD34+ cells per apheresis was 8.76 x 10(6)/Kg (range 1.83-27.87). None of the patients developed side effects which required hospitalization. All patients enrolled successively received HDC as consolidation treatment. High risk patients received one and metastatic patients two HDC with PBPC reinfusion. All patients obtained a complete engraftment. No significant differences between high-risk and metastatic patients were observed. CONCLUSIONS: Our study suggests that the combination of Docetaxel, Epirubicin, and G-CSF is feasible, safe and efficient outpatient mobilizing treatment for patients with breast cancer receiving HDC.

Acute hyperglycemia and acute hyperinsulinemia decrease plasma fibrinolytic activity and increase plasminogen activator inhibitor type 1 in the rat.

Decreased plasma fibrinolysis may contribute to accelerated atherothrombosis in diabetes. To observe whether hyperglycemia and hyperinsulinemia, common findings in type 2 diabetes, acutely affect plasma fibrinolysis in vivo, we evaluated plasma fibrinolysis (lysis of fibrin plates, free PAI-1 activity and t-PA activity) in the rat after a hyperglycemic euinsulinemic clamp (n=8), an euglycemic hyperinsulinemic clamp (n=7) or a saline infusion (n=15). Plasma fibrinolytic activity was sharply reduced after both the hyperglycemic and hyperinsulinemic clamps as compared to the respective controls (mean lysis areas on the fibrin plate, 139+/-21 vs. 323+/-30 mm2, p<0.001; 78+/-27 vs. 312+/-27 mm2 p<0.001, respectively). Plasma PAI-1 activity was greater after both hyperglycemic and hyperinsulinemic clamps as compared to saline infusion (6.6+/-2.6 vs. 1.6+/-0.6 IU/ml, p<0.001; 26+/-4 vs. 1.3+/-0.7 IU/ml, p<0.0001, respectively). Plasma t-PA activity was significantly reduced both after the hyperglycemic (0.36+/-0.15 vs. 2.17+/-0.18 IU/ml in controls, p<0.001) and the hyperinsulinemic (0.3+/-0.1 vs. 2.3+/-0.3 IU/ml in control, p<0.001) clamps. These data show that in vivo both acute hyperglycemia and acute hyperinsulinemia can decrease plasma fibrinolytic potential and that this is due to increased plasma PAI-1 and decreased free t-PA activities.

A phase I-II study of oral doxifluridine plus radiotherapy in radiosensitive tumors of the pelvic region.

AIMS AND BACKGROUND: Fluoropyrimidines have shown synergic effects in combination with radiotherapy in several tumor types. Doxifluridine is a novel 5-fluorouracil (5-FU) prodrug which is transformed into 5-FU in neoplastic tissue. This would imply enhancement of radiotherapy by 5-FU in neoplastic tissue, where the drug is concentrated higher than in surrounding healthy tissues. METHODS: A phase I-II study was carried out on 10 patients with radiosensitive tumors of the pelvic area (4 uterine carcinomas). Escalating doses of oral doxifluridine were administered in combination with standard radiotherapy to assess the efficacy and toxicity profile of the combined treatment. The 9 evaluable patients (3 groups of 3 patients each) received oral doxifluridine, at daily doses of 500, 750, or 1000 mg, for 6 consecutive weeks in combination with a standard (1.8-2.0 Gy) dose of radiotherapy. Assessment of physical and laboratory parameters was made at baseline, then weekly up to the end of the treatment and at follow-up. RESULTS: At the final evaluation, one patient with a diagnosis of uterine carcinoma showed a complete response that lasted 10 weeks. One patient had a partial response, and 7 patients had no change. The most frequent adverse events were gastrointestinal: 27 episodes of mild to moderate nausea/vomiting and diarrhea. Three patients complained of severe diarrhea of 5-7 days duration: all patients spontaneously recovered. There were no significant changes in laboratory or clinical parameters, and no serious toxicity requiring reduction or interruption of the radiotherapy. CONCLUSIONS: The maximum tolerated dose of oral doxifluridine in combination with standard radiotherpay was assessed at 1000 mg/patient/day (equivalent to 36-38 g monthly, previously reported as mTD in phase I studies).