Generation of hiPSCs Nkx2.5-eGFP reporter cell line for isolation of human cardiac progenitor cells

Choroby układu sercowo-naczyniowego (CVD) są poważnym wyzwaniem dla dzisiejszej medycyny i przyczyniają się do największej liczby zgonów na świecie. Obiecującym narzędziem do odbudowy mięśnia sercowego mogą być kardiomiocyty wyprowadzone z ludzkich indukowanych komórek macierzystych (hiPSCs). hiPSCs są otrzymywane na drodze reprogramowania komórek somatycznych pobranych bezpośrednio od pacjenta. Dane komórki można zróżnicować in vitro do różnych typów komórek, w tym do sercowych komórek progenitorowych (CPC) i kardiomiocytów (hiPSCs-CM). CPC oferują nowe podejście w leczeniu CVD z powodu zachowania ich multipotencji i zdolności do proliferacji. Wstępne różnicowanie hiPSCs do CPC pozwala zredukować ryzyko wystąpienia arytmii możliwej po przeszczepie hiPSCs-CM oraz stworzyć in vivo odpowiednie mikrośrodowisko złożone z różnych typów komórek. Izolacja ludzkich CPC jest utrudniona z powodu braku odpowiednich markerów powierzchniowych jednoznacznie definiujących dana pulę komórek. Natomiast zarówno komórki prekursorowe z pierwszego jak i drugiego pola sercowego cechują się ekspresja genu NKX2.5.W przedstawionej pracy wyprowadzono reporterową linię hiPSCs NKX2.5-eGFP do izolacji ludzkich CPC w oparciu o ekspresję NKX2.5 podczas różnicowania hiPSCs do kardiomiocytów. Do wprowadzenia sekwencji eGFP do lokus NKX2.5 w hiPSCs skorzystano z metody do precyzyjnej edycji genomu CRISPR/Cas9. W celu podwyższenia wydajności tej procedury opracowano protokół z zastosowaniem nokodazolu – związku dorbnocząsteczkowego zwiększającego efektywność CRISPR/Cas9. Wyprowadzone klony hiPSCs Nkx2.5-eGFP zróżnicowano w kierunku sercowej mezodermy i obserwowano ekspresję eGFP począwszy od dnia 6 tego procesu. Wykonano również częściową charakterystykę wyprowadzonej linii hiPSCs NKX2.5-eGFP pod kątem ekspresji markerów pluripotencji. Przeprowadzone doświadczenia stanowią ważny krok do opracowania optymalnych warunków hodowli ludzkich CPC oraz wydajnych protokołów ich różnicowania.Cardiovascular diseases (CVD) pose a reasonable challenge for modern medicine and contribute to the highest death rate in the world. A promising way to regenerate damaged heart muscle is to use cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs). hiPSCs are stem cells obtained by reprogramming somatic cells taken directly from a patient. They demonstrate capability to differentiate in vitro into different cell types including cardiac progenitor cells (CPC) and cardiomyocytes (hiPSCs-CM). CPC offer a novel approach in treating CVD because of their multipotency and retained ability to proliferate. Preliminary differentiation of hiPSCs to CPC allows to reduce arythmogenic risk possible after implantation of hiPSCs-CM in vivo and create a suitable microenvironment for heart regeneration. Isolation of human CPC is challenging due to the lack of appropriate cell surface markers defining these cells. Based on previous publications, NKX2.5 is a master gene manifested in the progenitor cells creating both the first heart field and the second heart field. In the present study a NKX2.5-eGFP reporter cell line was created to isolate human CPC. To introduce eGFP-encoding sequences into NKX2.5 gene CRISPR/Cas9 tool for precise genome editing was used. A two-step protocol to increase CRISPR/Cas9-mediated knock-in rate was established, using nocodazole, a small molecule increasing efficiency of homologous recombination. Derived hiPSCs clones were differentiated to cardiac mesoderm and green fluorescence in early CPC was observed starting from day 6. The hiPSCs NKX2.5-eGFP line was also partially characterized by expression of pluripotency markers. Obtained results constitute an important step towards generation of efficient methods for human CPC culture and differentiation

Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant

DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies

Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

Summary: Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits

Angiotensin II receptor blocker intake associates with reduced markers of inflammatory activation and decreased mortality in patients with cardiovascular comorbidities and COVID-19 disease

Aims Patients with cardiovascular comorbidities have a significantly increased risk for a critical course of COVID-19. As the SARS-CoV2 virus enters cells via the angiotensin-converting enzyme receptor II (ACE2), drugs which interact with the renin angiotensin aldosterone system (RAAS) were suspected to influence disease severity. Methods and results We analyzed 1946 consecutive patients with cardiovascular comorbidities or hypertension enrolled in one of the largest European COVID-19 registries, the Lean European Open Survey on SARS-CoV-2 (LEOSS) registry. Here, we show that angiotensin II receptor blocker intake is associated with decreased mortality in patients with COVID-19 [OR 0.75 (95% CI 0,59-0.96; p = 0.013)]. This effect was mainly driven by patients, who presented in an early phase of COVID-19 at baseline [OR 0,64 (95% CI 0,43-0,96; p = 0.029)]. Kaplan-Meier analysis revealed a significantly lower incidence of death in patients on an angiotensin receptor blocker (ARB) (n = 33/318;10,4%) compared to patients using an angiotensin-converting enzyme inhibitor (ACEi) (n = 60/348;17,2%) or patients who received neither an ACE-inhibitor nor an ARB at baseline in the uncomplicated phase (n = 90/466; 19,3%; p<0.034). Patients taking an ARB were significantly less frequently reaching the mortality predicting threshold for leukocytes (p<0.001), neutrophils (p = 0.002) and the inflammatory markers CRP (p = 0.021), procalcitonin (p = 0.001) and IL-6 (p = 0.049). ACE2 expression levels in human lung samples were not altered in patients taking RAAS modulators. Conclusion These data suggest a beneficial effect of ARBs on disease severity in patients with cardiovascular comorbidities and COVID-19, which is linked to dampened systemic inflammatory activity

Angiotensin II receptor blocker intake associates with reduced markers of inflammatory activation and decreased mortality in patients with cardiovascular comorbidities and COVID-19 disease

Aims Patients with cardiovascular comorbidities have a significantly increased risk for a critical course of COVID-19. As the SARS-CoV2 virus enters cells via the angiotensin-converting enzyme receptor II (ACE2), drugs which interact with the renin angiotensin aldosterone system (RAAS) were suspected to influence disease severity. Methods and results We analyzed 1946 consecutive patients with cardiovascular comorbidities or hypertension enrolled in one of the largest European COVID-19 registries, the Lean European Open Survey on SARS-CoV-2 (LEOSS) registry. Here, we show that angiotensin II receptor blocker intake is associated with decreased mortality in patients with COVID-19 [OR 0.75 (95% CI 0,59-0.96; p = 0.013)]. This effect was mainly driven by patients, who presented in an early phase of COVID-19 at baseline [OR 0,64 (95% CI 0,43-0,96; p = 0.029)]. Kaplan-Meier analysis revealed a significantly lower incidence of death in patients on an angiotensin receptor blocker (ARB) (n = 33/318;10,4%) compared to patients using an angiotensin-converting enzyme inhibitor (ACEi) (n = 60/348;17,2%) or patients who received neither an ACE-inhibitor nor an ARB at baseline in the uncomplicated phase (n = 90/466; 19,3%; p<0.034). Patients taking an ARB were significantly less frequently reaching the mortality predicting threshold for leukocytes (p<0.001), neutrophils (p = 0.002) and the inflammatory markers CRP (p = 0.021), procalcitonin (p = 0.001) and IL-6 (p = 0.049). ACE2 expression levels in human lung samples were not altered in patients taking RAAS modulators. Conclusion These data suggest a beneficial effect of ARBs on disease severity in patients with cardiovascular comorbidities and COVID-19, which is linked to dampened systemic inflammatory activity

The SARS-CoV-2 main protease M-pro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (M-pro) cleaves NEMO, the essential modulator of nuclear factor-kappa B. By ablating NEMO, M-pro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the M-pro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19. A novel study led by scientists in Lubeck, Germany, shows that SARS-CoV-2-infected brain endothelial cells undergo cell death due to the cleavage of NEMO by the viral protease M-pro, potentially causing cerebral COVID-19 and 'long COVID' symptoms