A Critical Analysis of the Available In Vitro and Ex Vivo Methods to Study Retinal Angiogenesis.

Angiogenesis is a biological process with a central role in retinal diseases. The choice of the ideal method to study angiogenesis, particularly in the retina, remains a problem. Angiogenesis can be assessed through in vitro and in vivo studies. In spite of inherent limitations, in vitro studies are faster, easier to perform and quantify, and typically less expensive and allow the study of isolated angiogenesis steps. We performed a systematic review of PubMed searching for original articles that applied in vitro or ex vivo angiogenic retinal assays until May 2017, presenting the available assays and discussing their applicability, advantages, and disadvantages. Most of the studies evaluated migration, proliferation, and tube formation of endothelial cells in response to inhibitory or stimulatory compounds. Other aspects of angiogenesis were studied by assessing cell permeability, adhesion, or apoptosis, as well as by implementing organotypic models of the retina. Emphasis is placed on how the methods are applied and how they can contribute to retinal angiogenesis comprehension. We also discuss how to choose the best cell culture to implement these methods. When applied together, in vitro and ex vivo studies constitute a powerful tool to improve retinal angiogenesis knowledge. This review provides support for researchers to better select the most suitable protocols in this field.

Distinct right ventricle remodeling in response to pressure overload in the rat.

Pulmonary arterial hypertension (PAH), the most serious chronic disorder of the pulmonary circulation, is characterized by pulmonary vasoconstriction and remodeling, resulting in increased afterload on the right ventricle (RV). In fact, RV function is the main determinant of prognosis in PAH. The most frequently used experimental models of PAH include monocrotaline- and chronic hypoxia-induced PAH, which primarily affect the pulmonary circulation. Alternatively, pulmonary artery banding (PAB) can be performed to achieve RV overload without affecting the pulmonary vasculature, allowing researchers to determine the RV-specific effects of their drugs/interventions. In this work, using two different degrees of pulmonary artery constriction, we characterize, in full detail, PAB-induced adaptive and maladaptive remodeling of the RV at 3 wk after PAB surgery. Our results show that application of a mild constriction resulted in adaptive hypertrophy of the RV, with preserved systolic and diastolic function, while application of a severe constriction resulted in maladaptive hypertrophy, with chamber dilation and systolic and diastolic dysfunction up to the isolated cardiomyocyte level. By applying two different degrees of constriction, we describe, for the first time, a reliable and short-duration PAB model in which RV adaptation can be distinguished at 3 wk after surgery. We characterize, in full detail, structural and functional changes of the RV in its response to moderate and severe constriction, allowing researchers to better study RV physiology and transition to dysfunction and failure, as well as to determine the effects of new therapies.

Worse cardiac remodeling in response to pressure overload in type 2 diabetes mellitus.

BACKGROUND: Diabetic cardiomyopathy is characterized by cardiac structural and functional abnormalities. Additionally, chronic pressure overload conditions are highly prevalent amongst diabetic population and this association leads to a more severe myocardial impairment. The differences in myocardial pathophysiology between type 1 and type 2 diabetes mellitus (DM) still remain to be clarified. Thus, we aimed to investigate biventricular structural and functional changes promoted by the two types of DM and the impact of concomitant chronic pressure overload. METHODS: Wistar rats were injected with streptozotocin (Type 1 DM, T1DM) or fed with a hypercaloric diet (Type 2 DM, T2DM). Pressure overload was imposed in DM animals by aortic constriction and after 5weeks of DM the cardiac function and structure were evaluated. RESULTS: Both types of DM promoted hypertrophy, increased fibrosis and advanced glycation end-products deposition, in the two ventricles. Interestingly, the induced myocardial alterations were distinct. While T1DM stimulated a pronounced hypertrophy and extracellular matrix remodeling, T2DM induced functional impairment. The negative impact of the association of DM with aortic constriction was more pronounced in T2DM, promoting impaired function and increased stiffness, particularly in the right ventricle. CONCLUSIONS: Our study demonstrated that the two types of diabetes induce distinct cardiac alterations per se or when combined with chronic pressure overload. T1DM promoted a more extensive remodeling in cardiac structure while T2DM significantly impaired ventricular function. The impact of pressure overload was more notorious in T2DM as observed by worse myocardial remodeling, suggesting a higher susceptibility to the deleterious effects of chronic pressure overload, namely hypertension, among this diabetic population.

S100A1: a major player in cardiovascular performance.

Calcium cycling is a major determinant of cardiac function. S100A1 is the most abundant member of the calcium-binding S100 protein family in myocardial tissue. S100A1 interacts with a variety of calcium regulatory proteins such as SERCA2a, ryanodine receptors, L-type calcium channels and Na(+)/Ca(2+) exchangers, thus enhancing calcium cycling. Aside from this major function, S100A1 has an important role in energy balance, myofilament sliding, myofilament calcium sensibility, titin-actin interaction, apoptosis and cardiac remodeling. Apart from its properties regarding cardiomyocytes, S100A1 is also important in vessel relaxation and angiogenesis. S100A1 potentiates cardiac function thus increasing the cardiomyocytes' functional reserve; this is an important feature in heart failure. In fact, S100A1 seems to normalize cardiac function after myocardial infarction. Also, S100A1 is essential in the acute response to adrenergic stimulation. Gene therapy experiments show promising results, although further studies are still needed to reach clinical practice. In this review, we aim to describe the molecular basis and regulatory function of S100A1, exploring its interactions with a myriad of target proteins. We also explore its functional effects on systolic and diastolic function as well as its acute actions. Finally, we discuss S100A1 gene therapy and its progression so far.

Intraventricular pressure gradients in heart failure.

The aim of the present study was to characterize intraventricular pressure gradients (IVPGs) in an animal model of chronic heart failure. New Zealand rabbits were treated with doxorubicin (heart failure group, n=5) or saline (control group, n=5) and instrumented with pressure catheters placed in the apex and outflow-tract of left ventricle (LV) and with sonomicrometer crystals placed in the apex and base of the LV free wall. In heart failure animals, ventricular filling was delayed and slower when compared with control animals. Moreover, the physiological nonuniformity observed between apical and basal segments in normal hearts was abolished in failing hearts. Simultaneously, physiological IVPGs observed during normal ventricular filling were entirely lost in heart failure animals. During ventricular emptying physiological nonuniformity between apical and basal segments observed in control animals was also abolished in heart failure animals. In failing hearts minimal length occurred later and almost at same time both in apical and in basal myocardial segments. Simultaneously, the characteristic IVPG pattern observed in healthy hearts during systole, which promotes ventricular emptying, was not observed in failing hearts. The present study showed that diastolic IVPGs, a marker of normal ventricular filling, and systolic IVPGs, a marker of normal ventricular emptying, are abolished in heart failure.

Synergistic impact of endurance training and intermittent hypobaric hypoxia on cardiac function and mitochondrial energetic and signaling.

BACKGROUND: Intermittent hypobaric-hypoxia (IHH) and endurance-training (ET) are cardioprotective strategies against stress-stimuli. Mitochondrial modulation appears to be an important step of the process. This study aimed to analyze whether a combination of these approaches provides additive or synergistic effects improving heart-mitochondrial and cardiac-function. METHODS: Two-sets of rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1 h/day/5 weeks treadmill-running), hypoxic-sedentary (HS, 6000 m, 5h/day/5 weeks) and hypoxic-exercised (HE) to study overall cardiac and mitochondrial function. In vitro cardiac mitochondrial oxygen consumption and transmembrane potential were evaluated. OXPHOS subunits and ANT protein content were semi-quantified by Western blotting. HIF-1α, VEGF, VEGF-R1 VEGF-R2, BNP, SERCA2a and PLB expressions were measured by qRT-PCR and cardiac function was characterized by echocardiography and hemodynamic parameters. RESULTS: Respiratory control ratio (RCR) increased in NE, HS and HE vs. NS. Susceptibility to anoxia/reoxygenation-induced dysfunction decreased in NE, HS and HE vs. NS. HS decreased mitochondrial complex-I and -II subunits; however HE completely reverted the decreased content in complex-II subunits. ANT increased in HE. HE presented normalized ventricular-arterial coupling (Ea) and BNP myocardial levels and significantly improved myocardial performance as evaluated by increased cardiac output and normalization of the Tei index vs. HS CONCLUSION: Data demonstrates that IHH and ET confer cardiac mitochondria with a more resistant phenotype although without visible addictive effects at least under basal conditions. It is suggested that the combination of both strategies, although not additive, results into improved cardiac function.

Rodent models of heart failure: an updated review.

Heart failure (HF) is one of the major health and economic burdens worldwide, and its prevalence is continuously increasing. The study of HF requires reliable animal models to study the chronic changes and pharmacologic interventions in myocardial structure and function and to follow its progression toward HF. Indeed, during the past 40 years, basic and translational scientists have used small animal models to understand the pathophysiology of HF and find more efficient ways of preventing and managing patients suffering from congestive HF (CHF). Each species and each animal model has advantages and disadvantages, and the choice of one model over another should take them into account for a good experimental design. The aim of this review is to describe and highlight the advantages and drawbacks of some commonly used HF rodents models, including both non-genetically and genetically engineered models, with a specific subchapter concerning diastolic HF models.

Modulation of myocardial stiffness by ß-adrenergic stimulation--its role in normal and failing heart.

The acute effects of beta-adrenergic stimulation on myocardial stiffness were evaluated. New-Zealand white rabbits were treated with saline (control group) or doxorubicin to induce heart failure (HF) (DOXO-HF group). Effects of isoprenaline (10(-10)-10(-5) M), a non-selective beta-adrenergic agonist, were tested in papillary muscles from both groups. In the control group, the effects of isoprenaline were also evaluated in the presence of a damaged endocardial endothelium, atenolol (beta(1)-adrenoceptor antagonist), ICI-118551 (beta(2)-adrenoceptor antagonist), KT-5720 (PKA inhibitor), L-NNA (NO-synthase inhibitor), or indomethacin (cyclooxygenase inhibitor). Passive length-tension relations were constructed before and after adding isoprenaline (10(-5) M). In the control group, isoprenaline increased resting muscle length up to 1.017+/-0.006 L/L(max). Correction of resting muscle length to its initial value resulted in a 28.5+/-3.1 % decrease of resting tension, indicating decreased muscle stiffness, as confirmed by the isoprenaline-induced right-downward shift of the passive length-tension relation. These effects were modulated by beta(1)- and beta(2)-adrenoceptors and PKA. In DOXO-HF group, the effect on myocardial stiffness was significantly decreased. We conclude that beta-adrenergic stimulation is a relevant mechanism of acute neurohumoral modulation of the diastolic function. Furthermore, this study clarifies the mechanisms by which myocardial stiffness is decreased.

Urotensin II-induced increase in myocardial distensibility is modulated by angiotensin II and endothelin-1.

Endogenous regulators, such as angiotensin-II (AngII), endothelin-1 (ET-1) and urotensin-II (U-II) are released from various cell types and their plasma levels are elevated in several cardiovascular diseases. The present study evaluated a potential crosstalk between these systems by investigating if the myocardial effects of U-II are modulated by AngII or ET-1. Effects of U-II (10(-8), 10(-7), 10(-6) M) were tested in rabbit papillary muscles in the absence and in the presence of losartan (selective AT(1) receptor antagonist), PD-145065 (nonselective ET-1 receptors antagonist), losartan plus PD-145065, AngII or ET-1. U-II promoted concentration-dependent negative inotropic and lusitropic effects that were abolished in all experimental conditions. Also, U-II increased resting muscle length up to 1.008+/-0.002 L/L(max). Correcting it to its initial value resulted in a 19.5+/-3.5 % decrease of resting tension, indicating increased muscle distensibility. This effect on muscle length was completely abolished in the presence of losartan and significantly attenuated by PD-145065 or losartan plus PD-145065. This effect was increased in the presence of AngII, resulting in a 27.5+/-3.9 % decrease of resting tension, but was unaffected by the presence of ET-1. This study demonstrated an interaction of the U-II system with the AngII and ET-1 systems in terms of regulation of systolic and diastolic function.

ETB2 receptor subtype stimulation relaxes the iris sphincter muscle.

Effects of ET(B) receptor stimulation and its subcellular pathways were evaluated in carbachol pre-contracted rabbit iris sphincter muscles (n=51). ET(B) stimulation with sarafotoxin (SRTX-c; 10(-10)-10(-6) M) was tested in the absence (n=7) or presence of 10(-5) M of: BQ-788 (ET(B2) receptor antagonist; n=6), L-NA (NOS inhibitor; n=7) or indomethacin (cyclooxygenase inhibitor; n=10). Effects of ET(B) stimulation by endothelin-1 (ET-1; 10(-10)-10(-7) M) in the presence of an ET(A) receptor antagonist (BQ-123; 10(-5) M; n=7) and of ET(B1) stimulation by IRL-1620 (10(-10)-10(-7) M; n=7) were also tested. Finally, the effects of SRTX-c (10(-9)-10(-7) M) in electric field stimulation (EFS) contraction were evaluated (n=7). ET(B) receptor stimulation by SRTX-c or ET-1 in presence of BQ-123 promoted a concentration-dependent relaxation of the rabbit iris sphincter muscle by 10.8+/-2.0% and 9.4+/-1.8%, respectively. This effect was blocked by BQ-788 (-2.3+/-2.0 %), L-NA (4.5+/-2.3 %) or indomethacin (2.3+/-2.9 %). Selective ET(B1) stimulation by IRL-1620 did not relax the iris sphincter muscle (0.9+/-5.4 %). EFS elicited contraction was not altered by SRTX-c. In conclusion, ET(B) receptor stimulation relaxes the carbachol precontracted iris sphincter muscle, an effect that is mediated by the ET(B2) receptor subtype, through NO and the release of prostaglandins.

Effects of TNF-alpha blockade in monocrotaline-induced pulmonary hypertension.

INTRODUCTION: TNF-alpha blockade in ischemic heart failure is still the subject of debate since clinical trials show conflicting results. However, its benefit in heart failure secondary to pulmonary hypertension has yet to be determined. It has been reported that transgenic rats overexpressing TNF-alpha develop pulmonary hypertension. The aim of this study was to assess the morphologic and hemodynamic effects of administration of an anti-TNF-alpha monoclonal antibody (etanercept) in rats with monocrotaline (MCT)-induced pulmonary hypertension. METHODS: Adult Wistar rats were injected with MCT (60 mg/Kg sc), or vehicle only (day 0). Beginning one day later, the animals were randomly treated with etanercept (ETC, 0.03 mg/Kg sc, three times a week) or with a similar volume of vehicle. The study thus had four groups: Ctrl (n = 6), Ctrl + ETC (n = 6), MCT (n = 6) and MCT + ETC (n = 6). On days 22-23, the rats were instrumented to record right ventricular systolic and end-diastolic pressures, dP/dtmax and tau. At the end of each experiment the heart and lungs were weighed. RESULTS AND CONCLUSIONS: Chronic administration of etanercept induced only a slight increase in relaxation velocity, with no effect on other hemodynamic parameters, including pulmonary hypertension, and no reduction in right ventricular hypertrophy. These results suggest that etanercept does not lead to a significant improvement in heart failure secondary to pulmonary hypertension.

The obestatin/ghrelin system as a novel regulatory mechanism of iris muscle contraction.

PURPOSE: To evaluate obestatin and ghrelin effects on iris muscle contraction. MATERIALS AND METHODS: Obestatin (10(-5) M) or ghrelin (10(-5) M) were tested on two consecutive carbachol-or epinephrine-elicited contractions of iris rabbit sphincter or dilator muscles. Ghrelin and obestatin effects on iris muscles basal tension were also tested, and their effects on iris sphincter EFS-elicited contraction were evaluated. RESULTS: Compared with the first, tension of the second carbachol-induced contraction of the iris sphincter decreased 11.5+/-5.5% in the vehicle group, increased 19.0+/-10.2% in presence of obestatin, and remained unchanged by ghrelin. Epinephrine-induced contractions were not affected by obestatin or ghrelin. EFS-elicited contractions were decreased 9.3+/-3.2% by ghrelin. Basal tension of the iris sphincter decreased 21.7+/-3.7% in presence of ghrelin (10(-5) M), while that of the dilator decreased 14.1+/-5.0% in presence of obestatin (10(-5) M). CONCLUSION: This study suggests that obestatin potentiates the cholinergic contraction of the iris sphincter and relaxes the iris dilator muscles.

Nitric oxide and prostaglandins - important players in endothelin-1 induced myocardial distensibility.

This study investigated whether endothelin (ET)-1-induced increase in myocardial distensibility is preserved in heart failure (HF) and whether it is modulated by nitric oxide (NO) and prostaglandins. New Zealand white rabbits were treated with doxorubicin (1 mg/kg, intravenously twice a week for 8 weeks, DOX-HF group) or saline (control group). Effects of ET-1 (0.1, 1, 10 nM) were tested in papillary muscles from the DOX-HF group and a control group in the presence of: i) intact endocardial endothelium (EE); ii) damaged EE; iii) N(G)-nitro-L-arginine (L-NNA; NO synthase inhibitor), and iv) indomethacin (INDO; cyclooxygenase inhibitor). In the presence of an intact EE, ET-1 promoted concentration-dependent positive inotropic and lusitropic effects that were maintained after damaging the EE, in the presence of L-NNA or INDO and in the DOX-HF Group. ET-1 reduced resting tension at the end of the isometric twitch (increased diastolic distensibility) by 3.2+/-1.3 %, 6.0+/-1.6 % and 8.8+/-2.7 % (at 0.1, 1 and 10 nM, respectively), in muscles with intact EE, effect that was completely abolished after damaging EE, in the presence of L-NNA or INDO or in the DOX-HF Group. This study demonstrated that the increase in myocardial distensibility induced by ET-1 is absent in HF and is dependent of NO and prostaglandin release.

Cardiac remodeling and dysfunction in nephrotic syndrome.

There is an increased incidence of heart disease in patients with chronic nephrotic syndrome (NS), which may be attributable to the malnutrition and activated inflammatory state accompanying the sustained proteinuria. In this study, we evaluated renal function, cardiac morphometry, contractile function, and myocardial gene expression in the established puromycin aminonucleoside nephrosis rat model of NS. Two weeks after aminonucleoside injection, there was massive proteinuria, decreased creatinine clearance, and a negative sodium balance. Skeletal and cardiac muscle atrophy was present and was accompanied by impaired left ventricular (LV) hemodynamic function along with decreased contractile properties of isolated LV muscle strips. The expression of selected cytokines and proteins involved in calcium handling in myocardial tissue was evaluated by real time polymerase chain reaction. This revealed that the expression of interleukin-1beta, tumor necrosis factor-alpha, and phospholamban were elevated, whereas that of cardiac sarco(endo)plasmic reticulum calcium pump protein was decreased. We suggest that protein wasting and systemic inflammatory activation during NS contribute to cardiac remodeling and dysfunction.

Ghrelin as a novel locally produced relaxing peptide of the iris sphincter and dilator muscles.

Ghrelin is a recently described acylated peptide, which works as a somatosecretagogue and has described effects on the smooth, skeletal and cardiac muscle. We examined the production and effects of ghrelin on relaxation of the iris muscles. Contractile effects of 1-5 human ghrelin (frGhr, 10(-9)-6 x 10(-5)M) and 1-5 human des-octanoyl-ghrelin (d-frGhr; 10(-9)-6 x 10(-5)M) were tested on iris rabbit sphincter (n=11 frGhr; n=7 d-frGhr), dilator (n=6 frGhr; n=6 d-frGhr) and rat sphincter (n=6 frGhr; n=8 d-frGhr) precontracted muscles. On rabbit sphincter the effect of frGhr was also tested in presence of: i) L-NA (10(-5)M; n=7); ii) indomethacin (10(-5)M; n=7); iii) DLys(3)GHRP6 (10(-4)M; n=6); and iv) apamin+carybdotoxin (10(-6)M; n=6). Furthermore, on rabbit dilator the effect of frGhr was tested in presence of DLys(3)GHRP6 (10(-4)M; n=7). Finally, ghrelin mRNA production was assessed by "in situ" hybridization in Wistar rat eyes (n=8). In all muscles, frGhr promoted a concentration-dependent relaxation, maximal at 6 x 10(-5)M, 1.5-3 min after its addition, decreasing tension by 34.1+/-12.1%, 25.8+/-4.8% and 52.1+/-10.3% in the rabbit sphincter, dilator and rat sphincter, respectively. In the rabbit sphincter the relaxing effects of frGhr were: (i) enhanced in presence of DLys(3)GHRP6 (118.1+/-21.1%); (ii) blunted by indomethacin; and (iii) not altered by apamin+carybdotoxin (36.4+/-14.4%) or L-NA (52.4+/-11.4%). Relaxing effects of d-frGhr in rabbit (43.3+/-5.2%) and rat (77.1+/-15.3%) sphincter muscles were similar to those of frGhr. In rabbit dilator muscle, d-frGhr did not significantly alter active tension and the relaxing effect of frGhr was blunted by GHSR-1a blockage. Ghrelin mRNA was identified in iris posterior epithelium. In conclusion, ghrelin is a novel, locally produced, relaxing agent of iris dilator and sphincter muscles, an effect that is mediated by GHSR-1a in the former, but not in the latter. Furthermore, in the sphincter it seems to be mediated by prostaglandins, but not by NO or K(Ca) channels.

Differential right and left ventricular diastolic tolerance to acute afterload and NCX gene expression in Wistar rats.

This study evaluated right ventricular (RV) and left ventricular (LV) diastolic tolerance to afterload and SERCA2a, phospholamban and sodium-calcium exchanger (NCX) gene expression in Wistar rats. Time constant tau and end diastolic pressure-dimension relation (EDPDR) were analyzed in response to progressive RV or LV afterload elevations, induced by beat-to-beat pulmonary trunk or aortic root constrictions, respectively. Afterload elevations decreased LV- tau, but increased RV-tau. Whereas LV- tau analyzed the major course of pressure fall, RV- tau only assessed the last fourth. Furthermore, RV afterload elevations progressively upward shifted RV EDPDR, whilst LV afterload elevations did not change LV-EDPDR. SERCA2a and phospholamban mRNA were similar in both ventricles. NCX-mRNA was almost 50 % lower in RV than in LV. Left ventricular afterload elevations, therefore, accelerated the pressure fall and did not induce diastolic dysfunction, indicating high LV diastolic tolerance to afterload. On the contrary, RV afterload elevations decelerated the late RV pressure fall and induced diastolic dysfunction, indicating small RV diastolic tolerance to afterload. These results support previous findings relating NCX with late Ca(2+) reuptake, late relaxation and diastolic dysfunction.

Pattern of right ventricular pressure fall and its modulation by afterload.

Pattern of right ventricular pressure (RVP) fall and its afterload dependence were examined by analyzing ventricular pressure curves and corresponding pressure dP/dt phase planes obtained in both ventricles in the rat heart in situ. Time and value of dP/dt(min), and the time constant tau were measured at baseline and during variable RV afterload elevations, induced by beat-to-beat pulmonary trunk constrictions. RVP and left ventricular pressure (LVP) decays were divided into initial accelerative and subsequent decelerative phases separated by corresponding dP/dt(min). At baseline, LVP fall was decelerative during 4/5 of its course, whereas only 1/3 of RVP decay occurred in a decelerative fashion. During RV afterload elevations, the absolute value of RV-dP/dt(min) and RV-tau increased, whilst time to RV dP/dt(min) decreased. Concomitantly, the proportion of RVP decay following a decelerative course increased, so that in highly RV afterloaded heartbeats RVP fall became more similar to LVP fall. In conclusion, RVP and LVP decline have distinct patterns, their major portion being decelerative in the LV and accelerative in the RV. In the RV, dP/dt(min), tau and the proportional contribution of accelerative and decelerative phases for ventricular pressure fall are afterload-dependent. Consequently, tau evaluates a relatively much shorter segment of RVP than LVP fall.