Rap1A Modulates Store-Operated Calcium Entry in the Lung Endothelium: A Novel Mechanism Controlling NFAT-Mediated Vascular Inflammation and Permeability.

BACKGROUND: Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 (calcium release-activated calcium modulator 1) is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 (Ras-proximate-1) isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation. METHODS: The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches. RESULTS: We explored Ca2+ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 µmol/L BTP2 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide), 10 µmol/L GSK-7975A, and 5 µmol/L Gd3+. Whole-cell patch clamp measurements revealed enhanced Ca2+ release-activated Ca2+ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1 [C-X-C motif chemokine ligand 1], CXCL11 [C-X-C motif chemokine 11], CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1AiΔEC) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1AiΔEC mice. CONCLUSIONS: Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca2+ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions.

Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells.

Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.

RNA therapeutics to control fibrinolysis: review on applications in biology and medicine.

Regulation of fibrinolysis, the process that degrades blood clots, is pivotal in maintaining hemostasis. Dysregulation leads to thrombosis or excessive bleeding. Proteins in the fibrinolysis system include fibrinogen, coagulation factor XIII, plasminogen, tissue plasminogen activator, urokinase plasminogen activator, α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, plasminogen activator inhibitor-1, α2-macroglobulin, and others. While each of these is a potential therapeutic target for diseases, they lack effective or long-acting inhibitors. Rapid advances in RNA-based technologies are creating powerful tools to control the expression of proteins. RNA agents can be long-acting and tailored to either decrease or increase production of a specific protein. Advances in nucleic acid delivery, such as by lipid nanoparticles, have enabled the delivery of RNA to the liver, where most proteins of coagulation and fibrinolysis are produced. This review will summarize the classes of RNA that induce 1) inhibition of protein synthesis, including small interfering RNA and antisense oligonucleotides; 2) protein expression, including messenger RNA and self-amplifying RNA; and 3) gene editing for gene knockdown and precise editing. It will review specific examples of RNA therapies targeting proteins in the coagulation and fibrinolysis systems and comment on the wide range of opportunities for controlling fibrinolysis for biological applications and future therapeutics using state-of-the-art RNA therapies.

Suppressing upregulation of fibrinogen after polytrauma mitigates thrombosis in mice.

BACKGROUND: Polytrauma results in systemic inflammation and increased circulating fibrinogen, which increases the risk of microvascular and macrovascular thrombosis that contributes to secondary organ damage and venous thromboembolism (VTE). There are no clinically approved agents to prevent hyperfibrinogenemia after polytrauma. We hypothesized that preventing the increase in fibrinogen levels after polytrauma would suppress thrombosis. METHODS: Small-interfering ribonucleic acid (siRNA) against fibrinogen was encapsulated in lipid nanoparticles (siFibrinogen). Mice underwent a model of polytrauma and were then given varying doses of siFibrinogen, control siRNA, or no treatment. Fibrinogen was measured for 1 week via enxyme-linked immunosorbent assay (ELISA). To model postinjury VTE, the inferior vena cava was ligated 2 days after polytrauma in a portion of the mice. Thrombus weight was measured 48 hours after the inferior vena cava was ligated. RESULTS: Treatment with siFibrinogen prevented hyperfibrinogenemia after trauma without exacerbating the hypofibrinogenemic state that occurs in the acute injury period (1 hour). In treated groups, fibrinogen was significantly lower from 6 hours postinjury through the 7-day monitoring period. Maximal fibrinogen reduction was observed at 72 hours. Here, mice that received 2.0 mg/kg of siFibrinogen had 1% of normal values relative to untreated mice, and mice that received 1.0 or 0.5 mg/kg had 4%. Mice treated with siFibrinogen that underwent the postinjury VTE model had significantly reduced thrombus weight compared with control siRNA-treated animals. More notably, among all siFibrinogen treated mice, 12 of 18 were completely protected from thrombosis, compared with 0 of 9 displaying protection in the control group. CONCLUSION: The rise of fibrinogen and the size of thrombi after polytrauma can be mitigated via the administration of siRNA against fibrinogen. siFibrinogen represents a promising novel target for VTE prophylaxis posttrauma.

Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine.

In vivo delivery of mRNA is promising for the study of gene expression and the treatment of diseases. Lipid nanoparticles (LNPs) enable efficient delivery of mRNA constructs, but protein expression has been assumed to be limited to the liver. With specialized LNPs, delivery to extrahepatic tissue occurs in small animal models; however, it is unclear if global delivery of mRNA to all major organs is possible in humans because delivery may be affected by differences in innate immune response and relative organ size. Furthermore, limited studies with LNPs have been performed in large animal models, such as swine, due to their sensitivity to complement activation-related pseudoallergy (CARPA). In this study, we found that exogenous protein expression occurred in all major organs when swine were injected intravenously with a relatively low dose of mRNA encapsulated in a clinically relevant LNP formulation. Exogenous protein was detected in the liver, spleen, lung, heart, uterus, colon, stomach, kidney, small intestine, and brain of the swine without inducing CARPA. Furthermore, protein expression was detected in the bone marrow, including megakaryocytes, hematopoietic stem cells, and granulocytes, and in circulating white blood cells and platelets. These results show that nearly all major organs contain exogenous protein expression and are viable targets for mRNA therapies.

siRNA-mediated reduction of a circulating protein in swine using lipid nanoparticles.

Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through in vivo delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.

Regulation of hepatic inclusions and fibrinogen biogenesis by SEL1L-HRD1 ERAD.

Impaired secretion of an essential blood coagulation factor fibrinogen leads to hepatic fibrinogen storage disease (HFSD), characterized by the presence of fibrinogen-positive inclusion bodies and hypofibrinogenemia. However, the molecular mechanisms underlying the biogenesis of fibrinogen in the endoplasmic reticulum (ER) remain unexplored. Here we uncover a key role of SEL1L-HRD1 complex of ER-associated degradation (ERAD) in the formation of aberrant inclusion bodies, and the biogenesis of nascent fibrinogen protein complex in hepatocytes. Acute or chronic deficiency of SEL1L-HRD1 ERAD in the hepatocytes leads to the formation of hepatocellular inclusion bodies. Proteomics studies followed by biochemical assays reveal fibrinogen as a major component of the inclusion bodies. Mechanistically, we show that the degradation of misfolded endogenous fibrinogen Aα, Bß, and γ chains by SEL1L-HRD1 ERAD is indispensable for the formation of a functional fibrinogen complex in the ER. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD indeed degrades and thereby attenuates the pathogenicity of two disease-causing fibrinogen γ mutants. Together, this study demonstrates an essential role of SEL1L-HRD1 ERAD in fibrinogen biogenesis and provides insight into the pathogenesis of protein-misfolding diseases.