Hemioksygenaasi-1 sydän- ja verisuonitaudeissa

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.Sepelvaltimotautikohtaus ja sydämen vajaatoiminta ovat yleisiä sairastavuuden ja kuolleisuuden syitä. Sydäninfarktin hoidossa pyritään mahdollisimman varhaiseen pallolaajennus-, leikkaus- tai liuotushoitoon sydänlihaksen verenkierron palauttamiseksi ja infarktin koon rajoittamiseksi. Sydäninfarktin seurauksena voi nykyisistä hoitomuodoista huolimatta kehittyä sydämen vajaatoiminta. Jotta sydäninfarktin ennustetta voitaisiin parantaa ja sydämen vajaatoiminnan kehittymistä ehkäistä, tarvitaan uusia keinoja sekä sydämen suojaamiseksi hapenpuutteen aiheuttamalta vauriolta että vaurioituneen sydänlihaksen paranemisen edistämiseksi. Hemioksygenaasi-1 (HO-1) on erityisesti reaktiivisten happiyhdisteiden aiheuttaman stressin seurauksena ilmentyvä, soluja suojaava entsyymi. HO-1 katalysoi hemin hajotuksen raudaksi, hiilimonoksidiksi (CO) ja biliverdiiniksi, joka edelleen pelkistetään bilirubiiniksi. HO-1:llä on osoitettu olevan mm. solukuolemaa, tulehdusreaktiota ja reaktiivisten happiyhdisteiden haitallista vaikutusta estäviä sekä verisuonten kasvua edistäviä (angiogeenisiä) vaikutuksia. Väitöstutkimuksessa selvitettiin HO-1:n kykyä estää ja korjata sydänlihasvauriota käyttämällä kokeellista sydäninfarktimallia ja eristettyjen sydänten iskemia/reperfuusio-mallia. Samoilla malleilla tutkittiin HO-1:n suojavaikutusmekanismeja. Tutkimuksessa selvitettiin myös HO-1 geenin polymorfioiden ja HO-1 plasmapitoisuuksien yhteyttä potilaiden ennusteeseen kriittisesti sairailla tehohoitoa vaativilla potilailla, joista osa oli sydänpotilaita. Havaitsimme kokeellisessa sydäninfarktimallissa, että HO-1:n ilmentyminen lisääntyi sydäninfarktin seurauksena erityisesti infarktialueella, infarktin reuna-alueella ja verisuonten seinämissä. HO-1 proteiinia havaittiin infarktin reuna-alueen sydänlihassolujen sarkoplasmakalvoston lisäksi niiden päätelevyissä, mikä voi viitata HO-1:n rytmihäiriöitä estävään vaikutukseen. Tutkittaessa HO-1:n ja CO:n vaikutuksia tarkemmin, havaitsimme, että HO-1 ja CO edesauttoivat infarktin paranemista ja sydänlihasvaurion korjaamista mm. lisäämällä verisuonien uudismuodostusta (neovaskularisaatiota) infarktialueella. HO-1:n ja CO:n vaikutukset välittyivät kuitenkin eri mekanismeilla. CO lisäsi vaskulogeneesiä ja sydänlihassolujen uudismuodostusta lisäämällä c-kit-positiivisten kantasolujen ilmaantumista infarktialueelle ja angiogeenisten tekijöiden (Hif-1a, SDF-1a ja VEGF-B) ilmentymistä infarktialueella. HO-1 puolestaan lisäsi infarktialueella angiogeneesiä, mahdollisesti SDF-1a:n välityksellä. Lisäksi HO-1 suojasi sydäntä vähentämällä sydänlihassolujen ohjelmoitua solukuolemaa. Infarktivaurion paranemisen varhaisvaiheessa HO-1 myös lisäsi infarktin reuna-alueen sydänlihassolujen jakautumista. HO-1 vähensi myös verisuonia ympäröivää sidekudosmuodostusta ja kollageeni I:n ilmentymistä. Osa HO-1:n suojavaikutuksista välittyi CO:n kautta, kuten solukuolemaa estävä vaikutus. CO:lla voi kuitenkin olla myös epäedullisia vaikutuksia sydämessä, sillä se lisäsi tärkeimpien soluväliaineen osien, kuten kollageeni I:n, III:n ja fibronektiinin ilmentymistä. Eristetyissä sydämissä HO-1:n lisääminen ennen hapenpuutetta paransi sen jälkeistä sydämen toimintaa ja esti reperfuusion aiheuttaman kammiovärinän. HO-1:n sydäntä suojaava vaikutus iskemia/reperfuusio-mallissa näyttäisi välittyvän osittain konneksiini 43:n kautta. Tutkimuksessa havaittiin myös, että HO-1 plasmapitoisuus oli selvästi koholla kaikilla tehohoitoa vaativilla potilailla, myös sydänpotilailla. HO-1 plasmapitoisuudet olivat yhteydessä elinhäiriöiden asteeseen ja sairauden vaikeusasteeseen. HO-1 plasmapitoisuudet olivat myös korkeammat potilailla, jotka kuolivat teho-osastolla tai sairaalassa verrattuna eloonjääneisiin. Neljän ensimmäisen tehohoitovuorokauden aikana mitattu korkein HO-1 plasmapitoisuus oli itsenäinen sairaalakuolleisuuden riskitekijä. Lisäksi havaitsimme, että HO-1 polymorfiat säätelevät HO-1 plasmapitoisuutta. HO-1 -413T/GT(L)/+99C haplotyyppi oli yhteydessä matalampiin HO-1 plasmapitoisuuksiin ja matalampaan monielinhäiriön insidenssiin. HO-1 polymorfioilla ei ollut yhteyttä kuolleisuuteen. Väitöskirjassa todettiin, että HO-1:n määrä lisääntyy stressitilanteissa sekä eläinmalleissa että kriittisesti sairailla potilailla. On mahdollista, että HO-1:tä tai sen reaktiotuotteita voidaan tulevaisuudessa käyttää hoitokokeiluissa hapenpuutteen aiheuttaman sydänvaurion hoidossa, sillä HO-1 ja CO säätelivät tällaisen sydänvaurion paranemis- ja korjausprosessia kokeellisessa infarktimallissa. Lisäksi HO-1 suojasi sydäntä iskemia/reperfuusio-vauriolta ja säilytti sydämen toimintakyvyn hapenpuutteen jälkeen. Tehohoitopotilaiden HO-1 plasmapitoisuus oli sitä suurempi mitä suurempi oli elinhäiriöiden aste, taudin vaikeusaste ja kuolleisuus ja voisi siten toimia näiden potilaiden sairauden vakavuuden ja ennusteen osoittajana

Unfavorable Reduction in the Ratio of Endothelin B to A Receptors in Experimental 5/6 Nephrectomy and Adenine Models of Chronic Renal Insufficiency

Chronic renal insufficiency (CRI) is characterized by increased endothelin 1 (ET-1) synthesis. We studied rat kidney endothelin receptor A (ETA) and receptor B (ETB) expressions after 12 and 27 weeks of 5/6 nephrectomy, and after 12 weeks of 0.3% adenine diet, representing proteinuric and interstitial inflammation models of CRI, respectively. Uric acid and calcium-phosphate metabolism were modulated after 5/6 nephrectomy, while ETA blocker and calcimimetic were given with adenine. Endothelin receptor mRNA levels were measured using RT-qPCR and protein levels using autoradiography (5/6 nephrectomy) or ELISA (adenine model). Both 12 and 27 weeks after 5/6 nephrectomy, kidney cortex ETA protein was increased by similar to 60% without changes in ETB protein, and the ETB:ETA ratio was reduced. However, the ETB:ETA mRNA ratio did not change. In the adenine model, kidney ETA protein was reduced by similar to 70%, while ETB protein was suppressed by similar to 95%, and the ETB:ETA ratio was reduced by similar to 85%, both at the protein and mRNA levels. The additional interventions did not influence the observed reductions in the ETB:ETAratio. To conclude, unfavorable reduction in the ETB:ETA protein ratio was observed in two different models of CRI. Therefore, ETA blockade may be beneficial in a range of diseases that cause impaired kidney function.Peer reviewe

Development of circulating microRNA-based biomarkers for medical decision-making : a friendly reminder of what should NOT be done

Circulating cell-free microRNAs (miRNAs) represent a major reservoir for biomarker discovery. Unfortunately, their implementation in clinical practice is limited due to a profound lack of reproducibility. The great technical variability linked to major pre-analytical and analytical caveats makes the interpretation of circulating cell-free miRNA data challenging and leads to inconsistent findings. Additional efforts directed to standardization are fundamental. Several well-established protocols are currently used by independent groups worldwide. Nonetheless, there are some specific aspects in specimen collection and processing, sample handling, miRNA quantification, and data analysis that should be considered to ensure reproducibility of results. Here, we have addressed this challenge using an alternative approach. We have highlighted and discussed common pitfalls that negatively impact the robustness of circulating miRNA quantification and their application for clinical decision-making. Furthermore, we provide a checklist usable by investigators to facilitate and ensure the control of the whole miRNA quantification and analytical process. We expect that these recommendations improve the reproducibility of findings, and ultimately, facilitate the incorporation of circulating miRNA profiles into clinical practice as the next generation of disease biomarkers.Peer reviewe

Consensus guidelines for the validation of qRT-PCR assays in clinical research by the CardioRNA consortium

Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking. Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and in vitro diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium (www. cardiorna.eu). Their applicability encompasses all clinical areas.Peer reviewe

Heme oxygenase-1 repeat polymorphism in septic acute kidney injury

Acute kidney injury (AKI) is a syndrome that frequently affects the critically ill. Recently, an increased number of dinucleotide repeats in the HMOX1 gene were reported to associate with development of AKI in cardiac surgery. We aimed to test the replicability of this finding in a Finnish cohort of critically ill septic patients. This multicenter study was part of the national FINNAKI study. We genotyped 300 patients with severe AKI (KDIGO 2 or 3) and 353 controls without AKI (KDIGO 0) for the guanine-thymine (GTn) repeat in the promoter region of the HMOX1 gene. The allele calling was based on the number of repeats, the cut off being 27 repeats in the S-L (short to long) classification, and 27 and 34 repeats for the S-M-L2 (short to medium to very long) classification. The plasma concentrations of heme oxygenase-1 (HO-1) enzyme were measured on admission. The allele distribution in our patients was similar to that published previously, with peaks at 23 and 30 repeats. The S-allele increases AKI risk. An adjusted OR was 1.30 for each S-allele in an additive genetic model (95% CI 1.01-1.66; p = 0.041). Alleles with a repeat number greater than 34 were significantly associated with lower HO-1 concentration (pPeer reviewe

Association of endothelial and glycocalyx injury biomarkers with fluid administration, development of acute kidney injury, and 90-day mortality : data from the FINNAKI observational study

Abstract Background Injury to endothelium and glycocalyx predisposes to vascular leak, which may subsequently lead to increased fluid requirements and worse outcomes. In this post hoc study of the prospective multicenter observational Finnish Acute Kidney Injury (FINNAKI) cohort study conducted in 17 Finnish intensive care units, we studied the association of Syndecan-1 (SDC-1), Angiopoetin-2 (Ang-2), soluble thrombomodulin (sTM), vascular adhesion protein-1 (VAP-1) and interleukin-6 (IL-6) with fluid administration and balance among septic critical care patients and their association with development of acute kidney injury (AKI) and 90-day mortality. Results SDC-1, Ang-2, sTM, VAP-1 and IL-6 levels were measured at ICU admission from 619 patients with sepsis. VAP-1 decreased (p  12 h from ICU admission (AKI>12 h). They had higher sTM levels than patients without AKI, and after multivariable adjustment log, sTM level was associated with AKI>12 h with OR (95% CI) of 12.71 (2.96–54.67), p = 0.001). Ninety-day non-survivors (n = 180; 29.1%) had higher SDC-1 and sTM levels compared to survivors. After adjustment for known confounders, log SDC-1 (OR [95% CI] 2.13 [1.31–3.49], p = 0.002), log sTM (OR [95% CI] 7.35 [2.29–23.57], p < 0.001), and log Ang-2 (OR [95% CI] 2.47 [1.44–4.14], p = 0.001) associated with an increased risk for 90-day mortality. Finally, patients who had high levels of all three markers, namely, SDC-1, Ang-2 and sTM, had an adjusted OR of 5.61 (95% CI 2.67–11.79; p < 0.001) for 90-day mortality. Conclusions VAP-1 and IL-6 associated with fluid administration on the first ICU day. After adjusting for confounders, sTM was associated with development of AKI after 12 h from ICU admission. SDC-1, Ang-2 and sTM were independently associated with an increased risk for 90-day mortality

Moderate hyperuricaemia ameliorated kidney damage in a low-renin model of experimental renal insufficiency

Uric acid has promoted renal fibrosis and inflammation in experimental studies, but some studies have shown nephroprotective effects due to alleviated oxidative stress. We studied the influence of experimental hyperuricaemia in surgically 5/6 nephrectomized rats. Three weeks after subtotal nephrectomy or sham operation, the rats were allocated to control diet or 2.0% oxonic acid (uricase inhibitor) diet for 9 weeks. Then blood, urine and tissue samples were taken, and renal morphology and oxidative stress were examined. Inflammation and fibrosis were evaluated using immunohistochemistry and real-time PCR (RT-PCR). Remnant kidney rats ingesting normal or oxonic acid diet presented with similar to 60% reduction of creatinine clearance and suppressed plasma renin activity. Oxonic acid diet increased plasma uric acid levels by >80 mu mol/L. In remnant kidney rats, moderate hyperuricaemia decreased glomerulosclerosis, tubulointerstitial damage and kidney mast cell count, without influencing the fibrosis marker collagen I messenger RNA (mRNA) content. In both sham-operated and 5/6 nephrectomized rats, the mast cell product 11-epi-prostaglandin-F-2 alpha excretion to the urine and kidney tissue cyclooxygenase-2 (COX-2) levels were decreased. To conclude, hyperuricaemic remnant kidney rats displayed improved kidney morphology and reduced markers of oxidative stress and inflammation. Thus, moderately elevated plasma uric acid had beneficial effects on the kidney in this low-renin model of experimental renal insufficiency.Peer reviewe

GSK3β Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury

Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy

Inhibition of let-7c Regulates Cardiac Regeneration after Cryoinjury in Adult Zebrafish

The let-7c family of micro-RNAs (miRNAs) is expressed during embryonic development and plays an important role in cell differentiation. We have investigated the role of let-7c in heart regeneration after injury in adult zebrafish. let-7c antagomir or scramble injections were given at one day after cryoinjury (1 dpi). Tissue samples were collected at 7 dpi, 14 dpi and 28 dpi and cardiac function was assessed before cryoinjury, 1 dpi, 7 dpi, 14 dpi and 28 dpi. Inhibition of let-7c increased the rate of fibrinolysis, increased the number of proliferating cell nuclear antigen (PCNA) positive cardiomyocytes at 7 dpi and increased the expression of the epicardial marker raldh2 at 7 dpi. Additionally, cardiac function measured with echocardiography recovered slightly more rapidly after inhibition of let-7c. These results reveal a beneficial role of let-7c inhibition in adult zebrafish heart regeneration