Spectroscopic Signature of Red Blood Cells in a D-Galactose-Induced Accelerated Aging Model.

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.

Comparison of Pulmonary and Systemic NO- and PGI2-Dependent Endothelial Function in Diabetic Mice.

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (Kf,c) and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO2- were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO2- generation, and production of 6-keto-PGF1α were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression.

FT-IR- and Raman-based biochemical profiling of the early stage of pulmonary metastasis of breast cancer in mice.

The combination of FT-IR and Raman spectroscopies allowed the biochemical profiling of lungs in the early stage of pulmonary metastasis in the murine model of breast cancer. Histological staining was used as a reference. Raman spectroscopy was especially useful in the detection and semi-quantitative analysis of the vitamin A content in lung lipofibroblasts, whereas the IR technique provided semi-quantitative information on the contents of nucleic acids, carbohydrates including glycogen, and lipids as well as changes in the secondary structures of tissue proteins. Our spectroscopic results suggest that the early phase of metastasis in the lung is characterized by a decrease in the endogenous retinoid content in combination with a decrease in the content of glycogen and lipids.

A possible Fourier transform infrared-based plasma fingerprint of angiotensin-converting enzyme inhibitor-induced reversal of endothelial dysfunction in diabetic mice.

Angiotensin-converting enzyme inhibitors (ACE-I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)-based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased ß-sheet formation. Spectral markers at 3010, 1520/1238 cm-1 , representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE-I.

Activation of the nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (MNA) pathway in pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with inflammatory response but it is unknown whether it is associated with alterations in NNMT activity and MNA plasma concentration. Here we examined changes in NNMT-MNA pathway in PAH in rats and humans. METHODS: PAH in rats was induced by a single subcutaneous injection of MCT (60 mg/kg). Changes in NNMT activity in the lungs and liver (assessed as the rate of conversion of nicotinamide (NA) to MNA), changes in plasma concentration of MNA and its metabolites (analyzed by LC/MS) were analyzed in relation to PAH progression. PAH was characterized by right ventricular hypertrophy (gross morphology), cardiac dysfunction (by MRI), lung histopathology, lung ultrastructure, and ET-1 concentration in plasma. NO-dependent and PGI2-dependent function in isolated lungs was analyzed. In naive patients with idiopathic pulmonary hypertension (IPAH) characterized by hemodynamic and biochemical parameters MNA and its metabolites in plasma were also measured. RESULTS: MCT-injected rats developed hypertrophy and functional impairment of the right ventricle, hypertrophy of the pulmonary arteries, endothelial ultrastructural defects and a progressive increase in ET-1 plasma concentration-findings all consistent with PAH development. In isolated lung, NO-dependent regulation of hypoxic pulmonary vasoconstriction was impaired, while PGI2 production (6-keto-PGF1α) was increased. NNMT activity increased progressively in the liver and in the lungs following MCT injection, and NNMT response was associated with an increase in MNA and 6-keto-PGF1α concentration in plasma. In IPAH patients plasma concentration of MNA was elevated as compared with healthy controls. CONCLUSIONS: Progression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.

Plasma biomarkers of pulmonary hypertension identified by Fourier transform infrared spectroscopy and principal component analysis.

The main goal of this study was to find specific plasma spectral markers associated with pulmonary arterial hypertension (PAH) induced by monocrotaline injection in rats. FTIR was used to monitor biochemical changes in plasma caused by PAH as compared with the systemic hypertension induced by partial ligation on the left artery and with the control group. Both pathologies, systemic and pulmonary hypertension, induced a unique response in the biochemical content of plasma, mainly related to the composition and secondary structure of plasma proteins. For PAH, ß-pleated sheet components of plasma proteins were identified whereas the protein composition in systemic hypertension was dominated by unordered structures. In addition, a higher concentration of tyrosine-rich proteins was found in plasma in PAH than in systemic hypertension. The differences between both pathologies were identified also in terms of lipid composition/metabolism as well as in the content of RNA and glucose, suggesting that lipid peroxidation appears upon pulmonary hypertension development. In summary, this work demonstrates that FTIR spectroscopy supported by principal component analysis (PCA) has the potential to become a fast and non-destructive method for biochemical characterization of plasma that consequently could have a diagnostic significance in pulmonary hypertension.

Visualization of the biochemical markers of atherosclerotic plaque with the use of Raman, IR and AFM.

In this work, we describe a methodology to visualize the biochemical markers of atherosclerotic plaque in cross sections of brachiocephalic arteries (BCA) taken from ApoE/LDLR(-/-) mice. The approach of the visualization of the same area of atherosclerotic plaque with the use of Raman, IR and AFM imaging enables the parallel characterisation of various features of atherosclerotic plaques. This support to the histochemical staining is utilized mainly in studies on mice models of atherosclerotic plaques, where micro and sub-micro resolutions are required. This work presents the methodology of the measurement and visualization of plaque features important for atherosclerosis development and plaques vulnerability analysis. Label-free imaging of cholesterol, cholesteryl esters, remodeled media, heme, internal elastic lamina, fibrous cap and calcification provides additional knowledge to previously presented quantitative measurements of average plaque features. AFM imaging enhanced the results obtained with the use of vibrational microspectroscopies with additional topographical information of the sample. To the best of our knowledge, this is the first work which demonstrates that co-localized measurement of atherosclerotic plaque with Raman, IR and AFM imaging provides a comprehensive insight into the biochemical markers of atherosclerotic plaques, and can be used as an integrated approach to assess vulnerability of the plaque.

Running performance at high running velocities is impaired but V'O(2max) and peripheral endothelial function are preserved in IL-6⁻/⁻ mice.

It has been reported that IL-6 knockout mice (IL-6⁻/⁻) possess lower endurance capacity than wild type mice (WT), however the underlying mechanism is poorly understood. The aim of the present work was to examine whether reduced endurance running capacity in IL-6⁻/⁻ mice is linked to impaired maximal oxygen uptake (V'O(2max)), decreased glucose tolerance, endothelial dysfunction or other mechanisms. Maximal running velocity during incremental running to exhaustion was significantly lower in IL-6⁻/⁻ mice than in WT mice (13.00±0.97 m·min⁻¹ vs. 16.89±1.15 m·min⁻¹, P<0.02, respectively). Moreover, the time to exhaustion during running at 12 m·min⁻¹ in IL-6⁻/⁻ mice was significantly shorter (P<0.05) than in WT mice. V'O(2max) in IL-6⁻/⁻ (n = 20) amounting to 108.3±2.8 ml·kg⁻¹·min⁻¹ was similar as in WT mice (n = 22) amounting to 113.0±1.8 ml·kg⁻¹·min⁻¹, (P = 0.16). No difference in maximal COX activity between the IL-6⁻/⁻ and WT mice in m. soleus and m. gastrocnemius was found. Moreover, no impairment of peripheral endothelial function or glucose tolerance was found in IL-6⁻/⁻ mice. Surprisingly, plasma lactate concentration during running at 8 m·min⁻¹ as well at maximal running velocity in IL-6⁻/⁻ mice was significantly lower (P<0.01) than in WT mice. Interestingly, IL-6⁻/⁻ mice displayed important adaptive mechanisms including significantly lower oxygen cost of running at a given speed accompanied by lower expression of sarcoplasmic reticulum Ca²âº-ATPase and lower plasma lactate concentrations during running at submaximal and maximal running velocities. In conclusion, impaired endurance running capacity in IL-6⁻/⁻ mice could not be explained by reduced V'O(2max), endothelial dysfunction or impaired muscle oxidative capacity. Therefore, our results indicate that IL-6 cannot be regarded as a major regulator of exercise capacity but rather as a modulator of endurance performance. Furthermore, we identified important compensatory mechanism limiting reduced exercise performance in IL-6⁻/⁻ mice.

Secondary structure of proteins analyzed ex vivo in vascular wall in diabetic animals using FT-IR spectroscopy.

In recent years many methods for ex vivo tissue analysis or diagnosis of diseases have been applied, including infrared absorption spectroscopy. Fourier-transform infrared (FT-IR) absorption microspectroscopy allows the simultaneous monitoring of the content of various chemical compounds in tissues with both high selectivity and resolution. Imaging of tissue samples in very short time can be performed using a spectrometer equipped with a Focal Plane Array (FPA) detector. Additionally, a detection of minor components or subtle changes associated with the functional status of a tissue sample is possible when advanced methods of data analysis, such as chemometric techniques, are applied. Monitoring of secondary structures of proteins has already proved to be useful in the analysis of animal tissues in disease states. The aim of this work was to build a mathematical model based on FT-IR measurements for the prediction of alterations in the content of secondary structures of proteins analyzed by FT-IR in the vascular wall of diabetic animals. For that purpose a spectral database of proteins of known crystallography and secondary structures was assembled. Thirty-seven proteins were measured by means of two FT-IR techniques: transflection and Attenuated Total Reflectance (ATR). The obtained model was tested on cross-sections of rat tail, for which the content of proteins and their secondary structures was well characterized. Then, the model was applied for the detection of possible alterations in the secondary structures of proteins in the vascular wall of diabetic rats and mice. The obtained results suggest a prominent increase in E- and S-structures and a decrease in the content of H-structures in the vascular wall from diabetic mice and rats. FT-IR-based studies of secondary structures of proteins may be a novel approach to study complex processes ongoing in the vascular wall. The obtained results are satisfactory; however, the existing limitations of the method are also discussed.

3D confocal Raman imaging of endothelial cells and vascular wall: perspectives in analytical spectroscopy of biomedical research.

Raman imaging was used to illustrate heterogeneity of a single endothelial cell and the vascular wall sample. The spectral analysis allowed for exploring the complexity of the studied systems in three dimensions and defining the size, volume, shape and biochemical composition of cellular organelles. The ability to construct the 3D maps by a method that does not disrupt the spatial integrity of the cell provided a unique insight into biochemical architecture and cellular processes of endothelium and vascular wall. 3D Raman imaging may be considered as a new trend in analytical spectroscopy to be applied in biomedical research. This method has a potential to be used for medical imaging and diagnostic purposes together with other, already established, imaging techniques.

Antithrombotic properties of water-soluble carbon monoxide-releasing molecules.

OBJECTIVE: We compared the antithrombotic effects in vivo of 2 chemically different carbon monoxide-releasing molecules (CORM-A1 and CORM-3) on arterial and venous thrombus formation and on hemostatic parameters such as platelet activation, coagulation, and fibrinolysis. The hypotensive response to CORMs and their effects on whole blood gas analysis and blood cell count were also examined. METHODS AND RESULTS: CORM-A1 (10-30 µmol/kg, i.v.), in a dose-dependent fashion, significantly decreased weight of electrically induced thrombus in rats, whereas CORM-3 inhibited thrombosis only at the highest dose used (30 µmol/kg). CORM-A1 showed a direct and stronger inhibition of platelet aggregation than CORM-3 in healthy rats, both in vitro and in vivo. The antiaggregatory effect of CORM-A1, but not CORM-3, correlated positively with weight of the thrombus. Concentration of active plasminogen activator inhibitor-1 in plasma also decreased in response to CORM-A1, but not to CORM-3. Neither CORM-A1 nor CORM-3 had an effect on plasma concentration of active tissue plasminogen activator. CORM-3, but not CORM-A1, decreased the concentration of fibrinogen, fibrin generation, and prolonged prothrombin time. Similarly, laser-induced venous thrombosis observed intravitally via confocal system in green fluorescent protein mice was significantly decreased by CORMs. Although both CORM-A1 and CORM-3 (30 µmol/kg) decreased platelets accumulation in thrombus, only CORM-A1 (3-30 µmol/kg) inhibited platelet activation to phosphatidylserine on their surface. CONCLUSIONS: CORM-3 and CORM-A1 inhibited thrombosis in vivo, however CORM-A1, which slowly releases carbon monoxide, and displayed a relatively weak hypotensive effect had a more pronounced antithrombotic effect associated with a stronger inhibition of platelet aggregation associated with a decrease in active plasminogen activator inhibitor-1 concentration. In contrast, the fast CO releaser CORM-3 that displayed a more pronounced hypotensive effect inhibited thrombosis primarily through a decrease in fibrin generation, but had no direct influence on platelet aggregation and fibrynolysis.

Inhibition of platelet aggregation by carbon monoxide-releasing molecules (CO-RMs): comparison with NO donors.

Carbon monoxide (CO) and CO-releasing molecules (CO-RMs) inhibit platelet aggregation in vitro. Herein, we compare the anti-platelet action of CORM-3, which releases CO rapidly (t (½) 1 min), and CORM-A1, which slowly releases CO (t(½) = 21 min). The anti-platelet effects of NO donors with various kinetics of NO release were studied for comparison. The effects of CO-RMs and NO donors were analyzed in washed human platelets (WP), platelets rich plasma (PRP), or whole blood (WB) using aggregometry technique. CORM-3 and CORM-A1 inhibited platelet aggregation in human PRP, WP, or WB, in a concentration-dependent manner. In all three preparations, CORM-A1 was more potent than CORM-3. Inhibition of platelets aggregation by CORM-A1 was not significantly affected by a guanylate cyclase inhibitor (ODQ) and a phosphodiesterase-5 inhibitor, sildenafil. In contrast, inhibition of platelet aggregation by NO donors was more potent with a fast NO releaser (DEA-NO, t (½) = 2 min) than slow NO releasers such as PAPA-NO (t (½) = 15 min) or other slow NO donors. Predictably, the anti-platelet effect of DEA-NO and other NO donors was reversed by ODQ while potentiated by sildenafil. In contrast to NO donors which inhibit platelets proportionally to the kinetics of NO released via activation of soluble guanylate cyclase (sGC), the slow CO-releaser CORM-A1 is a superior anti-platelet agent as compared to CORM-3 which releases CO instantly. The anti-platelet action of CO-RMs does not involve sGC activation. Importantly, CORM-A1 or its derivatives representing the class of slow CO releasers display promising pharmacological profile as anti-platelet agents.

Preserved cardiomyocyte function and altered desmin pattern in transgenic mouse model of dilated cardiomyopathy.

Taking advantage of the unique model of slowly developing dilated cardiomyopathy in mice with cardiomyocyte-specific transgenic overexpression of activated Gαq protein (Tgαq*44 mice) we analyzed the contribution of the cardiomyocyte malfunction, fibrosis and cytoskeleton remodeling to the development of heart failure in this model. Left ventricular (LV) in vivo function, myocardial fibrosis, cytoskeletal proteins expression and distribution, Ca(2+) handling and contractile function of isolated cardiomyocytes were evaluated at the stages of the early, compensated, and late, decompensated heart failure in 4-, 12- and 14-month-old Tgαq*44 mice, respectively, and compared to age-matched wild-type FVB mice. In the 4-month-old Tgαq*44 mice significant myocardial fibrosis, moderate myocyte hypertrophy and increased expression of regularly arranged and homogenously distributed desmin accompanied by increased phosphorylation of desmin chaperone protein, αB-crystallin, were found. Cardiomyocyte shortening, Ca(2+) handling and LV function were not altered. At 12 and 14 months of age, Tgαq*44 mice displayed progressive deterioration of the LV function. The contractile performance of isolated myocytes was still preserved, and the amplitude of Ca(2+) transients was even increased probably due to impairment of Na(+)/Ca(2+) exchanger function, while fibrosis was more extensive than in younger mice. Moreover, substantial disarrangement of desmin distribution accompanied by decreasing phosphorylation of αB-crystallin appeared. In Tgαq*44 mice disarrangement of desmin, at least partly related to inadequate phosphorylation of αB-crystallin seems to be importantly involved in the progressive deterioration of contractile heart function.

Determination of endothelin-1 in rats using a high-performance liquid chromatography coupled to electrospray tandem mass spectrometry.

A sensitive and specific liquid chromatography tandem mass spectrometry method with electrospray ionization for the determination of endothelin-1 in rat plasma and lung effluents has been developed and validated. Detection was achieved by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer coupled to an Agilent 1100 LC system. The limit of detection and the limit of the quantification of ET-1 in matrix buffer was estimated at 40 pM and 1 nM, respectively. The precision and accuracy for both intra- and inter-day determination of the analyte ranged from 2.5% to 14.7% and from 104.2% to 113.3%, respectively. No significant relative matrix effect was observed. Stability of ET-1 established in a bench-top, autosampler, long-term storage stability as well as freeze/thaw cycles shown no significant degradation products in the samples. The results of the method validation indicated that this method is applicable for the determination of the ET-1 concentration in an effluent from the isolated lung preparation as well as in vivo in plasma samples to evaluate ET-1 as a potential biomarker of the progression of pulmonary endothelial dysfunction and pulmonary hypertension in rats induced by a monocrotaline injection.

On the mechanism of coronary vasodilation induced by angiotensin-(1-7) in the isolated guinea pig heart.

Various mechanisms have been postulated to be involved in angiotensin-(1-7)-induced endothelium-dependent vasodilation. Here, we characterized the vasodilator action of angiotensin-(1-7) in the isolated guinea pig heart. Angiotensin-(1-7) (1-10 nmol, bolus) induced dose-dependent increase in the coronary flow. The coronary vasodilation induced by angiotensin-(1-7) was significantly reduced by the nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME) (100 microM) and abolished by a B(2) receptor antagonist, icatibant (100 nM). Coronary vasodilation induced by bradykinin (3 pmol, bolus) was inhibited by L-NAME and icatibant to similar extent as that induced by angiotensin-(1-7). Neither the selective AT(2) angiotensin receptor antagonist, PD123319 (1 microM), nor the antagonist of a putative angiotensin-(1-7) receptors, [D-alanine-7]-angiotensin-(1-7) (A-779, 1 microM), influenced the response to angiotensin-(1-7). In conclusion, in the isolated guinea pig heart angiotensin-(1-7) induces coronary vasodilation that is mediated by endogenous bradykinin and subsequent stimulation of nitric oxide release through endothelial B(2) receptors. In contrast to other vascular beds, AT(2) angiotensin receptors and specific angiotensin-(1-7) receptors do not appear involved in angiotensin-(1-7)-induced coronary vasodilation in the isolated guinea pig heart.

[Endothelium pharmacology in pulmonary hypertension]. / Farmakologia sródblonka w nadci nieniu plucnym.

The dysfunction of lung endothelium is crucial in the development of pulmonary hypertension. Dysfunction of endothelial synthesis of prostacyclin (PGI2) and nitric oxide (NO) and increased activity of endothelin 1 (ET-1) are connected to the progress of the disease. In this review the authors describe three major mediators of pulmonary endothelium: NO, PGI2 and ET-1. Their role in pulmonary hypertension and possibilities of pharmacological modulation of their activity are also discussed.