Biotechnological approach to induce human fibroblast apoptosis using superparamagnetic iron oxide nanoparticles.

Cancer-Associated Fibroblasts (CAFs) contribute to tumour progression and have received significant attention as a therapeutic target. These cells produce growth factors, cytokines and chemokines, stimulating cancer cell proliferation and inhibiting their apoptosis. Recent advances in drug delivery have demonstrated a significant promise of iron oxide nanoparticles in clinics as theranostic agents, mainly due to their magnetic properties. Here, we designed superparamagnetic iron oxide nanoparticles (SPIONs) to induce apoptosis of human fibroblasts. SPIONs were synthesized via co-precipitation method and coated with sodium citrate (SPION_Cit). We assessed the intracellular uptake of SPIONs by human fibroblast cells, as well as their cytotoxicity and ability to induce thermal effects under the magnetic field. The efficiency and time of nanoparticle internalization were assessed by Prussian Blue staining, flow cytometry and transmission electron microscopy. SPIONs_Cit were detected in the cytoplasm of human fibroblasts 15 min after in vitro exposure, entering into cells mainly via endocytosis. Analyses through Cell Titer Blue assay, AnnexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) cellular staining demonstrated that concentrations below 8 × 10-2 mg/mL of SPIONs_Cit did not alter cell viability of human fibroblast. Furthermore, it was also demonstrated that SPIONs_Cit associated with alternating current magnetic field were able to induce hyperthermia and human fibroblast cell death in vitro, mainly through apoptosis (83.5%), activating caspase 8 (extrinsic apoptotic via) after a short exposure period. Collectively these findings suggest that our nanoplatform is biocompatible and can be used for therapeutic purposes in human biological systems, such as inducing apoptosis of CAFs.

The synthetic peptide LyeTxI-b derived from Lycosa erythrognatha spider venom is cytotoxic to U-87 MG glioblastoma cells.

Antimicrobial peptides present a broad spectrum of therapeutic applications, including their use as anticancer peptides. These peptides have as target microbial, normal, and cancerous cells. The oncological properties of these peptides may occur by membranolytic mechanisms or non-membranolytics. In this work, we demonstrate for the first time the cytotoxic effects of the cationic alpha-helical antimicrobial peptide LyeTx I-b on glioblastoma lineage U87-MG. The anticancer property of this peptide was associated with a membranolytic mechanism. Loss of membrane integrity occurred after incubation with the peptide for 15 min, as shown by trypan blue uptake, reduction of calcein-AM conversion, and LDH release. Morphological studies using scanning electron microscopy demonstrated disruption of the plasma membrane from cells treated with LyeTx I-b, including the formation of holes or pores. Transmission electron microscopy analyses showed swollen nuclei with mild DNA condensation, cell volume increase with an electron-lucent cytoplasm and organelle vacuolization, but without the rupture of nuclear or plasmatic membranes. Morphometric analyses revealed a high percentage of cells in necroptosis stages, followed by necrosis and apoptosis at lower levels. Necrostatin-1, a known inhibitor of necroptosis, partially protected the cells from the toxicity of the peptide in a concentration-dependent manner. Imaging flow cytometry confirmed that 59% of the cells underwent necroptosis after 3-h incubation with the peptide. It is noteworthy that LyeTx I-b showed only mild cytotoxicity against normal fibroblasts of human and monkey cell lines and low hemolytic activity in human erythrocytes. All data together point out the anticancer potential of this peptide.

Angiotensin-(1-7) receptor Mas is an essential modulator of extracellular matrix protein expression in the heart.

In this study we investigated the effects of genetic deletion of the Angiotensin-(1-7) receptor Mas or the Angiotensin II receptor AT(2) on the expression of specific extracellular matrix (ECM) proteins in atria, right ventricles and atrioventricular (AV) valves of neonatal and adult mice. Quantification of collagen types I, III and VI and fibronectin was performed using immunofluorescence-labeling and confocal microscopy. Picrosirius red staining was used for the histological assessment of the overall collagen distribution pattern. ECM proteins, metalloproteinases (MMP), ERK1/2 and p38 levels were quantified by western blot analysis. Gelatin zymography was used to evaluate the activity of MMP-2 and MMP-9. We observed that the relative levels of collagen types I and III and fibronectin are significantly higher in both the right ventricle and AV valves of neonatal Mas(-/-) mouse hearts (e.g., collagen type I: 85.28±6.66 vs 43.50±4.41 arbitrary units in the right ventricles of Mas(+/+) mice). Conversely, the level of collagen type VI was lower in the right ventricle and AV valves of Mas(-/-) mice. Adult Mas(-/-) mouse hearts presented similar patterns as observed in neonates. No significant differences in ECM protein level were detected in atria. Likewise, no changes in ECM levels were observed in AT(2) knockout mouse hearts. Although deletion of Mas induced a significant reduction in the level of the active form of MMP-2 in neonate hearts and a reduction of both MMP-2 and MMP-9 in adult Mas(-/-) mice, no significant differences were observed in MMP enzymatic activities when compared to controls. The levels of the active, phosphorylated forms of ERK1/2 and p38 were higher in hearts of both neonatal and adult Mas(-/-) mice. These observations suggest that Mas is involved in the selective expression of specific ECM proteins within both the ventricular myocardium and AV valves. The changes in the ECM profile may alter the connective tissue framework and contribute to the decreased cardiac performance observed in Mas(-/-) mice.

Indirect effects of oral tolerance improve wound healing in skin.

Tissue injury in adult mammalian skin frequently results in scarring while fetal mammalian skin heals with complete regeneration. Inflammatory reactions are among the factors thought to impair regeneration. Previous studies have shown that the injection of an immunologically tolerated protein blocks immune responses to unrelated antigens and is also able to inhibit inflammation in mice. This phenomenon, which we refer to as the indirect effects of oral tolerance, does not require the simultaneous injection of the tolerated antigen and the second antigen, and also occurs when the two antigens are given by separate routes of immunization. Herein, we investigated whether the i.p. injection of an orally tolerated antigen (ovalbumin, OVA) would inhibit inflammatory reactions at an incisional lesion and influence healing of adult mouse skin. In OVA-tolerant mice, the injection of OVA minutes before wounding altered inflammation: it reduced the numbers of mast cells, neutrophils, and lymphocytes but increased the number of macrophages around the lesion area. Tolerant mice also showed fewer myofibroblasts and reduced scar area. Furthermore, tolerant mice displayed a pattern of extracellular matrix deposition similar to that observed in intact skin, plus characteristics of regeneration, such as an increased deposition of fibronectin and tenascin-C. These observations suggest that the indirect effects of oral tolerance can alter the process of wound healing in skin and reduce scar formation.

Genetic deletion of the angiotensin-(1-7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria.

Angiotensin-(1-7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1-7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-beta mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney.

Angiotensin-(1-7) activates a tyrosine phosphatase and inhibits glucose-induced signalling in proximal tubular cells.

BACKGROUND: In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, angiotensin-(1-7) [Ang-(1-7)] blocks activation of MAPKs by angiotensin II. We studied the effect of Ang-(1-7) on signalling responses in LLC-PK(1) cells in normal (5 mM) or high (25 mM) glucose. METHODS: The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity was measured after immunoprecipitation, cell protein synthesis was determined by [(3)H]-leucine incorporation and transforming growth factor-beta1 (TGF-beta1), fibronectin and collagen IV were assayed by immunoblots and/or ELISA. RESULTS: High glucose stimulated p38 MAPK. This response was inhibited by Ang-(1-7) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-779. Ang-(1-7) increased SHP-1 activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(1-7) on high glucose-stimulated p38 MAPK. Ang-(1-7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta1. Conversely, Ang-(1-7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV. CONCLUSIONS: These data indicate that in proximal tubular cells, binding of Ang-(1-7) to the receptor Mas stimulates SHP-1, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(1-7) selectively inhibits glucose-stimulated protein synthesis and TGF-beta1. In diabetic nephropathy, Ang-(1-7) may partly counteract the profibrotic effects of high glucose.

Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection.

BACKGROUND: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. METHODS AND FINDINGS: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. CONCLUSION: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Impairment of in vitro and in vivo heart function in angiotensin-(1-7) receptor MAS knockout mice.

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas(-/-) mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4+/-0.09 versus 2.1+/-0.03 g in Mas(+/+) mice), +/-dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart.