Novel biomarkers and emerging tools to identify causal molecular pathways in hypertension and associated cardiovascular diseases

Hypertension (HT) is a modifiable risk factor for life-threatening cardiovascular diseases (CVDs) including coronary artery disease, heart failure or stroke. Despite significant progress in understanding of the pathophysiological mechanisms of the disease, the molecular pathways targeted by HT treatment still remain largely unchanged. This warrants the necessity for searching novel biomarkers, which are causally related to persistent high blood pressure (BP) and may be pharmacologically targeted. Data from large-scale biobanks, containing high-throughput genetic and biochemical data, such as OLINK and SomaScan-based proteomics or Nuclear Magnetic Resonance-based metabolomics, as well as novel analytical tools including Mendelian randomisation (MR) approach enabling genetic casual inference, may create new treatment opportunities for HT and related CVDs. MR analysis may constitute an additional proof for observational studies and facilitate selection of druggable targets for clinical testing and have been already used to nominate potentially causal biomarkers for HT and CVDs such as circulating glycine, branched-chain amino acids, insulin-like growth factor 1 or fibronectin 1. Using MR framework, genetic proxies for targets of already known drugs, such as statins, PCSK9 and ACE inhibitors, may additionally inform about potential side effects and eventually contribute to a more personalized medicine. Finally, genetic causal inference may disentangle independent, direct effects of correlated traits such as lipid classes or markers of inflammation on cardiovascular clinical outcomes such as atherosclerosis and HT. While several novel HT-targeting drugs are currently under clinical investigation (e.g. brain renin-angiotensin-aldosterone system inhibitors or endothelin-1 receptor antagonists), analysis of high-throughput proteomic and metabolomic data from well-powered studies may deliver novel druggable molecular targets for HT and associated CVDs

The role of microRNA-378a in the formation of choroidal microvasculature

Badania prowadzone na przestrzeni ostatnich lat wykazały istotny wpływ miR-378a na proces tworzenia nowych naczyń krwionośnych, zarówno w aspekcie fizjologicznym, jak i patologicznym. W celu zbadania udziału miRNA-378a w tworzeniu sieci małych naczyń, czyli mikrowaskulatury, jako model badawczy zastosowano tak zwany test naczyniówkowy (ang. Choroid Sprouting Assay.Over the past years, research has revealed prominent impact of miR-378a in physiological and pathological angiogenesis. In order to investigate the contribution of miR-378a to microvascular developement, Choroid Sprouting Assay was performed

The role of heme oxygenase-1 in the regulation of osteoclastogenesis and osteoclast activity

Oksygenaza hemowa-1 (HO-1), której funkcją metaboliczną jest degradacja hemu do biliwerdyny, tlenku węgla oraz żelaza, jest enzymem o silnych właściwościach przeciwzapalnych, antyoksydacyjnych, cytoprotekcyjnych i proangiogennych. Jej ochronna rola oraz proangiogenne działanie sprawiają, że HO-1 może być istotna w terapii chorób związanych z zapaleniem, sercowo-naczyniowych czy nowotworów. Takim schorzeniom często towarzyszyć może zachwianie homeostazy tkanki kostnej i nasilona aktywność osteoklastów, komórek kościogubnych. W świetle danych wskazujących na istotne znaczenie cytokin prozapalnych czy reaktywnych form tlenu w procesie osteoklastogenezy, potencjalny udział HO-1 także w regulacji procesów przebudowy tkanki kostnej jest prawdopodobny i mógłby sugerować wielopłaszczyznowe zastosowanie terapeutyczne tego enzymu. Celem pracy było więc zbadanie udziału HO-1 w regulacji tego procesu, z uwzględnieniem wpływu enzymu na prekursory osteoklastów, na wczesne etapy osteoklastogenezy oraz na aktywność dojrzałych osteoklastów. Jako model badawczy zastosowano myszy z brakiem genu hmox1 kodującego HO-1 (HO-1-/-) i myszy typu dzikiego (HO-1+/+) oraz linię mysich makrofagów, RAW264.7. Dodatkowo sprawdzano wpływ wyciszenia ekspresji HO-1 przy użyciu siRNA oraz stosowano farmakologiczne induktory HO-1. W niniejszej pracy wykazano, że niedobór HO-1 wpływa na odsetek oraz kondycję prekursorów osteoklastów w szpiku kostnym powodując, pomimo wzrostu odsetka populacji monocytów, istotny spadek odsetka makrofagów, który może być związany z nieznacznie zmniejszonym potencjałem proliferacyjnym przy braku HO-1. Po stymulacji czynnikiem M-CSF nie odnotowano jednak różnic w liczebności i żywotności ani produkcji RFT przez hodowane komórki szpiku kostnego pomiędzy grupami HO-1-/- oraz HO-1+/+, pomimo tendencji do obniżonego potencjału proliferacyjnego przy braku HO-1. Dalsze badania wykazały, że zarówno brak genu hmox1, jak i wyciszenie HO-1 w prekursorach makrofagowych, skutkuje zmniejszeniem potencjału różnicowania takich komórek do osteoklastów, gdyż zaobserwowano spadek aktywności enzymu TRAP, a także zmniejszoną ekspresję innych markerów osteoklastogenezy, NFAT-c1 i katepsyny K. Co ciekawe, hamującego efektu niedoboru HO-1 na osteoklastogenezę nie obserwowano, kiedy ekspresję HO-1 wyciszano we wczesnych etapach różnicowania (po stymulacji czynnikiem RANKL indukującym różnicowanie). Zanotowano wręcz nieznaczny wzrost ekspresji markera różnicowania NFAT-c1. Ponadto, wyniki badań in vivo wykazały wzrost poziomu enzymu TRAP w osoczu krwi myszy HO-1-/-, co sugeruje wzrost aktywności resorpcyjnej osteoklastów przy braku HO-1. Podsumowując, wykazano, że pomimo hamującego efektu niedoboru HO-1 w prekursorach makrofagowych na indukcję procesu osteoklastogenezy, różnicowanie i aktywność osteoklastów mogą być względnie zwiększone przy braku HO-1. Zgodnie z tym można wnioskować, że HO-1 jest istotny dla indukcji procesu osteoklastogenezy, ale działa hamująco na aktywność dojrzałych osteoklastów.Heme oxygenase-1 which functions as a catalyst of heme degradation producing carbon monoxide, biliverdin and ferrous ion has strong anti-inflammatory, antioxidant, cytoprotective and proangiogenic properties. Thanks to its protective role and proangiogenic function HO-1 may be useful in therapies of inflammatory, cardiovascular and cancer diseases. Such disorders often may be accompanied by imbalance in bone tissue homeostasis which is connected with an increased osteoclast activity. In the light of data showing the crucial role of proinflammatory cytokines and reactive oxygen species in the process of osteoclastogenesis, the potential involvement of HO-1 in the regulation of bone remodeling is possible and might suggest a multi-faceted therapeutic use of this enzyme. The aim of this project was to analyze the role of HO-1 in the regulation of osteoclastogenesis with taking into account the effect of the enzyme on osteoclast precursors, the early stages of osteoclastogenesis and the activity of mature osteoclasts. As an experimental model mice lacking hmox1 gene encoding HO-1 (HO-1-/-) or wild type mice (HO-1+/+) and also RAW264.7 murine macrophage cell line were used. Moreover, the effect of silencing of the HO-1 expression using siRNA or the pharmacological inducers of HO-1 were used. Research has demonstrated that HO-1 deficiency affects number and condition of osteoclast precursors in bone marrow resulting in, despite increased percentage of monocyte population, significant decrease in macrophage abundance which may be associated with slightly reduced proliferative potential in the absence of HO-1. However, after M-CSF stimulation no significant differences nor in macrophage abundance and survival, neither in reactive oxygen species production in bone marrow cells derived from HO-1+/+ or HO-1-/- mice were reported, despite the tendency to decreased proliferative potential in absence of HO-1. Further studies indicated that both the lack of hmox1 gene and HO-1 deficiency in macrophages results in their decreased differentiation potential to osteoclast, as reduced TRAP activity and lowered expression of osteoclastogenesis markers such as NFAT-c1 and cathepsin K was observed. Interestingly, an inhibitory effect of HO-1 deficiency on osteoclastogenesis was not reported when HO-1 expression was silenced during early stages of differentiation (after RANKL stimulation). Even a slight increased expression of osteoclast marker NFAT-c1 was noted. Moreover, in vivo studies revealed increased level of TRAP enzyme in the plasma of HO-1-/- mice, which suggests increased resorptive activity of osteoclasts in the HO-1 absence. To summarize, it was demonstrated that despite an inhibitory effect of HO-1 deficiency in osteoclast precursor macrophages on the induction of osteoclastogenesis, differentiation and activity of osteoclast may be relatively elevated in the HO-1 absence. According to this, it can be concluded that HO-1 is important for osteoclastogenesis induction, but it inhibits mature osteoclast activity

Silencing of Sphingosine kinase 1 Affects Maturation Pathways in Mouse Neonatal Cardiomyocytes

Sphingosine kinase-1 (Sphk1) and its product, sphingosine-1-phosphate (S1P) are important regulators of cardiac growth and function. Numerous studies have reported that Sphk1/S1P signaling is essential for embryonic cardiac development and promotes pathological cardiac hypertrophy in adulthood. However, no studies have addressed the role of Sphk1 in postnatal cardiomyocyte (CM) development so far. The present study aimed to assess the molecular mechanism(s) by which Sphk1 silencing might influence CMs development and hypertrophy in vitro. Neonatal mouse CMs were transfected with siRNA against Sphk1 or negative control, and subsequently treated with 1 µM angiotensin II (AngII) or a control buffer for 24 h. The results of RNASeq analysis revealed that diminished expression of Sphk1 significantly accelerated neonatal CM maturation by inhibiting cell proliferation and inducing developmental pathways in the stress (AngII-induced) conditions. Importantly, similar effects were observed in the control conditions. Enhanced maturation of Sphk1-lacking CMs was further confirmed by the upregulation of the physiological hypertrophy-related signaling pathway involving Akt and downstream glycogen synthase kinase 3 beta (Gsk3β) downregulation. In summary, we demonstrated that the Sphk1 silencing in neonatal mouse CMs facilitated their postnatal maturation in both physiological and stress conditions

Serum and vascular stiffness biomarkers associated with the severity of degenerative aortic valve stenosis and cardiovascular outcomes

Background: Although degenerative aortic valve stenosis (DAS) is the most prevalent growth-up congestive heart valve disease, still little known about relationships between DAS severity, vascular stiffness (VS), echocardiographic parameters, and serum biomarkers in patients undergoing transcatheter (TAVR) or surgical aortic valve replacement (SAVR). The objective of this study was to identify biomarkers associated with DAS severity, and those that are associated with cardiovascular death (CVD) and episodes of chronic heart failure (CHF) exacerbation. Methods: A total of 137 patients with initially moderate-to-severe DAS were prospectively evaluated for the relationship between DAS severity, baseline VS, and serum biomarkers (uPAR, GDF-15, Gal-3, IL-6Rα, ET-1, PCSK9, RANTES/CCL5, NT-proBNP, and hs-TnT), and were followed-up for 48 months. The prognostic significance of each variable for CVD and CHF risk was measured by hazard ratio of risk (HR), which was calculated by Cox’s proportional hazard model. Results: DAS severity showed correlations with IL-6Rα (r = 0.306, p 11.5 ng/mL increased the risk of CHF exacerbation episodes requiring hospital admission by 12%. Conclusions: Our study indicated that ET-1 and Gal-3 levels may be associated with the outcomes in patients with DAS