STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Comparative transcriptomic analysis reveals translationally relevant processes in mouse models of malaria

Recent initiatives to improve translation of findings from animal models to human disease have focussed on reproducibility but quantifying the relevance of animal models remains a challenge. Here, we use comparative transcriptomics of blood to evaluate the systemic host response and its concordance between humans with different clinical manifestations of malaria and five commonly used mouse models. Plasmodium yoelii 17XL infection of mice most closely reproduces the profile of gene expression changes seen in the major human severe malaria syndromes, accompanied by high parasite biomass, severe anemia, hyperlactatemia, and cerebral microvascular pathology. However, there is also considerable discordance of changes in gene expression between the different host species and across all models, indicating that the relevance of biological mechanisms of interest in each model should be assessed before conducting experiments. These data will aid the selection of appropriate models for translational malaria research, and the approach is generalizable to other disease models.

Computational investigation of blood cell transport in retinal microaneurysms

Microaneurysms (MAs) are one of the earliest clinically visible signs of diabetic retinopathy (DR). MA leakage or rupture may precipitate local pathology in the surrounding neural retina that impacts visual function. Thrombosis in MAs may affect their turnover time, an indicator associated with visual and anatomic outcomes in the diabetic eyes. In this work, we perform computational modeling of blood flow in microchannels containing various MAs to investigate the pathologies of MAs in DR. The particle-based model employed in this study can explicitly represent red blood cells (RBCs) and platelets as well as their interaction in the blood flow, a process that is very difficult to observe in vivo. Our simulations illustrate that while the main blood flow from the parent vessels can perfuse the entire lumen of MAs with small body-to-neck ratio (BNR), it can only perfuse part of the lumen in MAs with large BNR, particularly at a low hematocrit level, leading to possible hypoxic conditions inside MAs. We also quantify the impacts of the size of MAs, blood flow velocity, hematocrit and RBC stiffness and adhesion on the likelihood of platelets entering MAs as well as their residence time inside, two factors that are thought to be associated with thrombus formation in MAs. Our results show that enlarged MA size, increased blood velocity and hematocrit in the parent vessel of MAs as well as the RBC-RBC adhesion promote the migration of platelets into MAs and also prolong their residence time, thereby increasing the propensity of thrombosis within MAs. Overall, our work suggests that computational simulations using particle-based models can help to understand the microvascular pathology pertaining to MAs in DR and provide insights to stimulate and steer new experimental and computational studies in this area.

The SARS-CoV-2 main protease Mpro 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 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro 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 Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

AB0461 “SCLERODERMA-LIKE” PATTERN AS A PRESENTING FEATURE OF CONNECTIVE TISSUE DISEASES

Background:The role of capillaroscopy for early diagnosis of systemic sclerosis (SSc) is well-known and pathological capillaroscopic pattern is a component of the new set of criteria for SSc (EULAR/ACR 2013). While it is also known that similar microvascular changes i.e., “scleroderma-like” could be observed in other rheumatic diseases i.e., undifferentiated connective tissue disease (UCTD), overlap syndromes, systemic lupus erythematosus (SLE), etc., the data about the time of their appearance in other rheumatic diseases different from SSc are scarce.Objectives:The aim of the study was to evaluate the prevalence of capillaroscopic features of microangiopathy in Raynaud’s phenomenon (RP) patients at the time of their first referral to rheumatology setting.Methods:22 in- and outpatients were included in the study that were referred for consultation in our rheumatology unit in the last 6 months. Inclusion criteria were presence of RP at their first consultation or still unclear diagnosis. Presence of known rheumatic disease diagnosed at previous consultation with rheumatologist as well as signs of definite diagnosis SSc were exclusion criteria. All the patients underwent capillaroscopic examination with USB capillaroscope Dinolite (magnification 200x). Routine laboratory tests were ordered i.e., complete blood count, ESR, CRP, biochemistry as well as immunological tests. ANA test was performed in all patients while antibodies against extractable nuclear antigens, antiphospholipid antibodies or other tests were ordered depending on the clinical presentation and overall context. The patients signed an informed consent for participation of the study.Results:12 of the examined patients were diagnosed with primary RP and their capillaroscopic examination revealed absence of microangiopathy i.e., normal pattern or non-specific changes (mainly dilated capillaries). In 7 patients the final diagnosis was UCTD and 4 of them exhibited microvascular pathology i.e. “scleroderma-like” pattern, while in 3 cases the capillaroscopic findings were non-specific. Among other patients 1 case was diagnosed with prescleroderma with “early” phase “scleroderma” pattern (according to definition of Cutolo et al., 2000 (1)), 1 case was with onset of SLE (“scleroderma-like” pattern, active phase) and in one case the microvascular pathology that included single giant capillary loop no other signs of connective tissue disease were found and the final conclusion was “suspected secondary” RP with necessity for a regular follow-up.Conclusion:In conclusion, definite features of microvascular pathology, known as “scleroderma-like” capillaroscopic pattern, could be observed as an initial pathological feature in CTD different from SSc such as UCTD and SLE and the overall diagnosis should be made in the overall context. Capillaroscopy inherits high significance in patients with UCTD, in whom clinical presentation could be obscure in the beginning and identification of microvascular capillaroscopic pathology is among the crucial signs to support the diagnosis. Future studies are necessary to delineate the role of microvascular pathology for prediction of future evolution of UCTD.References:[1]Cutolo M, Sulli A, Pizzorni C AS. Nailfold videocapillaroscopy assessment of microvascular damage in systemic sclerosis. J Rheumatol. 2000;27(1):155–60.Disclosure of Interests:None declared.

Comparative transcriptomic analysis reveals translationally relevant processes in mouse models of malaria

Recent initiatives to improve translation of findings from animal models to human disease have focussed on reproducibility but quantifying the relevance of animal models remains a challenge. Here we use comparative transcriptomics of blood to evaluate the systemic host response and its concordance between humans with different clinical manifestations of malaria and five commonly used mouse models. Plasmodium yoelii 17XL infection of mice most closely reproduces the profile of gene expression changes seen in the major human severe malaria syndromes, accompanied by high parasite biomass, severe anemia, hyperlactatemia, and cerebral microvascular pathology. However, there is also considerable discordance of changes in gene expression between species and across all models, indicating that the relevance of biological mechanisms of interest in each model should be assessed before conducting experiments. Our data will aid selection of appropriate models for translational malaria research, and the approach is generalizable to other disease models.

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.