Pedro Melgar-Lesmes

Multifunctional polyurethane–urea nanoparticles to target and arrest inflamed vascular environment: A potential tool for cancer therapy and diagnosis

Activation of inflammatory pathways in endothelial cells contributes to tumour growth and progression in multiple human cancers. Cellular adhesion molecules are involved in leukocyte recruitment to the vascular inflammatory environment where it plays a critical role in angiogenesis, suppression of apoptosis, proliferation, invasion and metastasis. We describe here the development of streptavidin-coated polyurethane-urea nanoparticles as multifunctional nanocarriers for fluorescence imaging or targeting of the tumour environment to identify and arrest the vascular network irrigating the tumour tissue. The design of these multifunctional nanoparticles involves incorporating streptavidin to the nanoparticle polymeric matrix. The obtained nanoparticles are spherical and exhibit an average diameter of 70-74 nm. Streptavidin-coated nanoparticles spontaneously bind biotinylated antibodies against VCAM-1 and ICAM-1 which in vitro and in vivo specifically attached to inflamed endothelial cells. Indeed the incorporation of CBO-P11 (a specific inhibitor of the vascular endothelial growth factor proangiogenic and proinflammatory pathway) to these nanoparticles allows a targeted pharmacological effect thereby decreasing the proliferation only in inflamed endothelial cells. The multiple functionalisation strategy described here could be exploited for tumour diagnostics or targeted drug delivery to tumour vasculature with a good safety profile and an attractive array of possibilities for biomedical applications.

The hepatic apelin system: A new therapeutic target for liver disease

Apelin is a peptide that plays an important role in heart physiology and pathophysiology, inflammation, and angiogenesis. We evaluated whether the endogenous apelin system is involved in the pathogenesis of the hepatic remodeling and cardiovascular and renal complications occurring in advanced liver disease. The circulating levels of apelin, the messenger RNA (mRNA) and protein expression of apelin and apelin receptor, the immunohistological detection of apelin and apelin receptor, and the effects induced by the chronic administration of an apelin receptor antagonist on fibrosis and vessel density were evaluated in rats with cirrhosis and ascites and in control rats. The serum levels of apelin in patients with cirrhosis were also measured. Apelin levels were higher in rats with cirrhosis than in controls. Apelin mRNA showed a four‐fold rise only in hepatic tissue, but not in the lung, heart, or kidney of rats with cirrhosis. These animals also showed hepatic apelin receptor mRNA levels 300 times higher than controls. Apelin was highly expressed by stellate cells, whereas apelin receptor was overexpressed in the hepatic parenchyma of animals with cirrhosis. Rats with cirrhosis treated with the apelin receptor antagonist showed diminished hepatic fibrosis and vessel density, improved cardiovascular performance, and renal function and lost ascites. Human patients also showed a marked increase in apelin levels. Conclusion: The selective hepatic activation of the apelin system, together with the drop in fibrosis and neoangiogenesis and the improvement in cardiovascular and excretory function resulting from apelin receptor blockade, points to the hepatic apelin system as a novel therapeutic target in liver disease. (HEPATOLOGY 2008.)

Hypoxia and proinflammatory factors upregulate apelin receptor expression in human stellate cells and hepatocytes

Background The activation of the apelin receptor (APJ) plays a major role in both angiogenic and fibrogenic response to chronic liver injury. However, the mechanisms that govern the induction of APJ expression have not been clarified so far. Methods The regulation and the role of APJ in cultured human liver cells were investigated. Tissular expression of APJ and α-smooth muscle actin was analysed by immunocolocalisation in human cirrhotic liver and in control samples. mRNA and protein expression of APJ were analysed in two cell lines, LX-2 (as hepatic stellate cells, HSCs) and HepG2 (as hepatocytes), under hypoxic conditions or after exposure to proinflammatory or profibrogenic factors. Additionally, both hepatic cell lines were stimulated with apelin to assess cell survival and the expression of angiogenic factors. Results The APJ-positive signal was negligible in control livers. In contrast, APJ was highly expressed in HSCs and slightly expressed in hepatocytes of human cirrhotic liver. Sustained hypoxia and lipopolysaccharide stimulated the expression of APJ in LX-2 cells. Moreover, hypoxia, tumour necrosis factor α and angiotensin II induced the expression of APJ in HepG2 cells. Activation of APJ stimulated angiopoietin-1 expression and cell survival in LX-2 cells and, in turn, triggered the synthesis of vascular endothelial growth factor type A and platelet-derived growth factor-BB in HepG2 cells. Conclusions These results suggest that hypoxia and inflammatory factors could play a major role in the activation of the hepatic apelin system leading to angiogenic and fibroproliferative response occurring in chronic liver disease.

Prevention of Fibrosis Progression in CCl4-Treated Rats: Role of the Hepatic Endocannabinoid and Apelin Systems

Endocannabinoids behave as antifibrogenic agents by interacting with cannabinoid CB2 receptors, whereas the apelin (AP) system acts as a proangiogenic and profibrogenic mediator in the liver. This study assessed the effect of long-term stimulation of CB2 receptors or AP receptor (APJ) blockade on fibrosis progression in rats under a non-discontinued fibrosis induction program. The study was performed in control and CCl4-treated rats for 13 weeks. Fibrosis-induced rats received a CB2 receptor agonist (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) (1 mg/kg b.wt.), an APJ antagonist [Ala13]-apelin-13 sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala (F13A) (75 μg/kg b.wt.), or vehicle daily during the last 5 weeks of the CCl4 inhalation program. Mean arterial pressure (MAP), portal pressure (PP), hepatic collagen content, angiogenesis, cell infiltrate, and mRNA expression of a panel of fibrosis-related genes were measured in all animals. Fibrosis-induced rats showed increased hepatic collagen content, reduced MAP, portal hypertension, and increased expression of the assessed messengers in comparison with control rats. However, fibrotic rats treated with either AM1241 or F13A had reduced hepatic collagen content, improved MAP and PP, ameliorated cell viability, and reduced angiogenesis and cell infiltrate compared with untreated fibrotic rats. These results were associated with attenuated induction of platelet-derived growth factor receptor β, α-smooth muscle actin, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinase. CB2 receptor stimulation or APJ blockade prevents fibrosis progression in CCl4-treated rats. The mechanisms underlying these phenomena are coincident despite the marked dissimilarities between the CB2 and APJ signaling pathways, thus opening new avenues for preventing fibrosis progression in liver diseases.

Apelin Mediates the Induction of Profibrogenic Genes in Human Hepatic Stellate Cells

UNLABELLED Apelin is a peptide with relevant functions in angiogenesis and inflammation. Recent studies have demonstrated that apelin is overexpressed in hepatic stellate cells (HSCs) of cirrhotic rats. Moreover, patients with cirrhosis show high circulating levels of this peptide. We evaluated the role of endogenous apelin system in fibrogenesis-related gene induction in human HSCs. Messenger expression and immunolocalization of apelin were analyzed in human cirrhotic liver and in control samples. Apelin expression was analyzed in a human HSC line (LX-2) under hypoxic conditions or in the presence of proinflammatory or profibrogenic stimuli. LX-2 cells were stimulated with apelin, and a selected profile of fibrogenesis-related genes was quantified. In vivo inactivation of apelin was analyzed in the liver of fibrotic rats after administrating specific blockers of the molecules triggering apelin induction. Apelin was overexpressed in HSCs from human cirrhotic liver. Neither hypoxia nor proinflammatory substances induced the expression of apelin in LX-2. By contrast, both profibrogenic molecules angiotensin II (AII) and endothelin-1 (ET-1) enhanced apelin expression in these cells. Apelin increased the synthesis of collagen-I and platelet-derived growth factor receptor β (PDGFRβ) in LX-2. AII and ET-1 stimulated collagen-I and PDGFRβ expression, and this induction was drastically reduced when apelin receptor was blocked in these cells. In accordance, AII or ET-1 receptor antagonists reduced the hepatic synthesis of apelin, collagen-I, and PDGFRβ in fibrotic rats. CONCLUSIONS apelin mediates some of the fibrogenic effects triggered by AII and ET-1, thus suggesting that apelin could be an important mediator of fibrogenesis in human liver disease.

Vascular endothelial growth factor and angiopoietin-2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats

Background and aims: The extent and molecular mechanisms governing plasma extravasation and formation of ascites in cirrhosis are unknown. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are endogenous substances with powerful vascular permeability effects. We assessed regional blood flow, vascular leakage, mRNA and tissular expression of VEGF-A and Ang-2 and vascular permeability following VEGF receptor 2 blockade in control and cirrhotic rats to define the vascular territories showing altered vascular permeability in cirrhosis and to determine whether VEGF-A and Ang-2 are involved in this phenomenon. Methods: Arterial blood flow was analysed with the coloured microsphere method. Vascular leakage was measured and visualised with the dye Evan’s Blue and colloidal carbon techniques, respectively. VEGF-A and Ang-2 expression were determined by real-time polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. The effect on vascular permeability induced by VEGFR2 blockade was assessed by administration of the receptor inhibitor SU11248. Results: Arterial blood flow was increased in the mesentery, pancreas and small intestine but not in the kidney and spleen of cirrhotic rats as compared to controls. Increased vascular leakage was observed in the mesentery and liver, where colloidal carbon spread from microvessels to the adjacent fibrotic tracts. Increased hepatic and mesenteric expression of VEGF-A and Ang-2 was found in cirrhotic rats as compared to controls. Blockade of VEGFR2 markedly reduced hepatic and mesenteric vascular leakage in cirrhotic rats. Conclusions: Enhanced endothelial permeability is restricted to the hepatic and mesenteric vascular beds in cirrhotic rats with ascites and VEGF-A and Ang-2 are key factors in the signalling pathways regulating this dysfunction.

Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats

Background and aim The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. Methods Cirrhosis was induced in rats by CCl4 inhalation. Lymphatic drainage was evaluated using fluorescent lymphangiography. Expression of endothelial nitric oxide synthase (eNOS) was measured in primary lymphatic endothelial cells (LyECs). Inhibition of eNOS activity in cirrhotic rats with ascites (CH) was carried out by L-NG-methyl-L-arginine (L-NMMA) treatment (0.5 mg/kg/day). Results The (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. Conclusions The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

Adenoviral dominant-negative soluble PDGFRβ improves hepatic collagen, systemic hemodynamics, and portal pressure in fibrotic rats

BACKGROUND & AIMS Platelet-derived growth factor (PDGF) is the most potent stimulus for proliferation and migration of stellate cells. PDGF receptor β (PDGFRβ) expression is an important phenotypic change in myofibroblastic cells that mediates proliferation and chemotaxis. Here we analyzed the relationship between PDGFRβ expression, hemodynamic deterioration, and fibrosis in CCl(4)-treated rats. Thereafter, we investigated the effects produced by an adenovirus encoding a dominant-negative soluble PDGFRβ (sPDGFRβ) on hemodynamic parameters, PDGFRβ signaling pathway, and fibrosis. METHODS Mean arterial pressure, portal pressure, PDGFRβ mRNA expression, and hepatic collagen were assessed in 6 controls and 21 rats induced to hepatic fibrosis/cirrhosis. Next, 30 fibrotic rats were randomized into three groups receiving iv saline and an adenovirus encoding for sPDGFRβ or β-galactosidase. After 7days, mean arterial pressure, portal pressure, serum sPDGFRβ, and hepatic collagen were measured. RESULTS CCl(4)-treated animals for 18weeks showed a significantly higher increase in PDGFRβ mRNA compared to those treated for 13weeks and control rats. In CCl(4)-treated rats, the fibrous tissue area ranged from moderate to severe fibrosis. A direct relationship between the degree of fibrosis, hemodynamic changes, and PDGFRβ expression was observed. Fibrotic rats transduced with the adenovirus encoding sPDGFRβ showed increased mean arterial pressure, decreased portal pressure, lower activation of the PDGFRβ signaling pathway, and reduced hepatic collagen than fibrotic rats receiving β-galactosidase or saline. CONCLUSIONS PDGFRβ activation closely correlates with hemodynamic disorders and increased fibrosis in CCl(4)-treated rats. Adenoviral dominant negative soluble PDGFRβ improved fibrosis. As a result, the hemodynamic abnormalities were ameliorated.

Polyurethane and polyurea nanoparticles based on polyoxyethylene castor oil derivative surfactant suitable for endovascular applications.

The design of new, safe and effective nanotherapeutic systems is an important challenge for the researchers in the nanotechnology area. This study describes the formation of biocompatible polyurethane and polyurea nanoparticles based on polyoxyethylene castor oil derivative surfactant formed from O/W nano-emulsions by polymerization at the droplet interfaces in systems composed by aqueous solution/Kolliphor(®) ELP/medium chain triglyceride suitable for intravenous administration. Initial nano-emulsions incorporating highly hydrophilic materials were prepared by the phase inversion composition (PIC) method. After polymerization, nanoparticles with a small particle diameter (25-55 nm) and low polydispersity index were obtained. Parameters such as concentration of monomer, O/S weight ratio as well as the polymerization temperature were crucial to achieve a correct formation of these nanoparticles. Moreover, FT-IR studies showed the full conversion of the monomer to polyurethane and polyurea polymers. Likewise the involvement of the surfactant in the polymerization process through their nucleophilic groups to form the polymeric matrix was demonstrated. This could mean a first step in the development of biocompatible systems formulated with polyoxyethylene castor oil derivative surfactants. In addition, haemolysis and cell viability assays evidenced the good biocompatibility of KELP polyurethane and polyurea nanoparticles thus indicating the potential of these nanosystems as promising drug carriers.

Factors Involved in Extracellular Matrix Turnover in Human Derived Cardiomyocytes

Background: The molecular mechanisms by which myocardial ischemia translates into ventricular remodeling remain unclear. Methods: We investigated whether hypoxia and proinflammatory cytokines are specific inducers of remodeling signals in an in vitro model of cultured adult human ventricular myocytes (AC16 cells). Results:Hypoxia modified the ratio of matrix remodeling factors by increasing the aminoterminal propeptide of type III procollagen (PIIINP) and reducing tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) secretion in AC16 cells. These effects, however, were not associated with either modifications in expression of matrix metalloproteinase type 2, collagen-I or metalloproteinase activity. Hypoxia does, actually increase the production of the cardiac antifibrogenic growth factors, Apelin and VEGF, through an Hypoxia Inducible Factor type 1-dependent mechanism. Concerning proinflammatory signaling pathways, IL1β emerged as a powerful inducer of matrix turnover, since it significantly enhanced PIIINP, TIMP-1 and hyaluronic acid production and increased metalloproteinase activity. In contrast, TNFα did not modify matrix turnover but markedly induced the production of Apelin and VEGF. Conclusion: Hypoxia and increased TNFα activity likely exert cardioprotective actions by activating the cardiac antifibrogenic factors Apelin and VEGF. In contrast, IL1β is a strong promoter of interstitial collagen remodeling that may contribute to ventricular dilation and heart failure in the ischemic myocardium.