Spike protein of SARS-CoV-2 activates macrophages and contributes to induction of acute lung inflammation in male mice.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in mediating viral entry into host cells. However, whether it contributes to pulmonary hyperinflammation in patients with coronavirus disease 2019 is not well known. In this study, we developed a spike protein-pseudotyped (Spp) lentivirus with the proper tropism of the SARS-CoV-2 spike protein on the surface and determined the distribution of the Spp lentivirus in wild-type C57BL/6J male mice that received an intravenous injection of the virus. Lentiviruses with vesicular stomatitis virus glycoprotein (VSV-G) or with a deletion of the receptor-binding domain (RBD) in the spike protein [Spp (∆RBD)] were used as controls. Two hours postinfection (hpi), there were 27-75 times more viral burden from Spp lentivirus in the lungs than in other organs; there were also about 3-5 times more viral burden from Spp lentivirus than from VSV-G lentivirus in the lungs, liver, kidney, and spleen. Deletion of RBD diminished viral loads in the lungs but not in the heart. Acute pneumonia was observed in animals 24 hpi. Spp lentivirus was mainly found in SPC+ and LDLR+ pneumocytes and macrophages in the lungs. IL6, IL10, CD80, and PPAR-γ were quickly upregulated in response to infection in the lungs as well as in macrophage-like RAW264.7 cells. Furthermore, forced expression of the spike protein in RAW264.7 cells significantly increased the mRNA levels of the same panel of inflammatory factors. Our results demonstrated that the spike protein of SARS-CoV-2 confers the main point of viral entry into the lungs and can induce cellular pathology. Our data also indicate that an alternative ACE2-independent viral entry pathway may be recruited in the heart and aorta.

Effects of emodin, a plant-derived anthraquinone, on TGF-ß1-induced cardiac fibroblast activation and function.

Cardiac fibrosis accompanies a number of pathological conditions and results in altered myocardial structure, biomechanical properties and function. The signaling networks leading to fibrosis are complex, contributing to the general lack of progress in identifying effective therapeutic approaches to prevent or reverse this condition. Several studies have shown protective effects of emodin, a plant-derived anthraquinone, in animal models of fibrosis. A number of questions remain regarding the mechanisms whereby emodin impacts fibrosis. Transforming growth factor beta 1 (TGF-ß1) is a potent stimulus of fibrosis and fibroblast activation. In the present study, experiments were performed to evaluate the effects of emodin on activation and function of cardiac fibroblasts following treatment with TGF-ß1. We demonstrate that emodin attenuates TGF-ß1-induced fibroblast activation and collagen accumulation in vitro. Emodin also inhibits activation of several canonical (SMAD2/3) and noncanonical (Erk1/2) TGF-ß signaling pathways, while activating the p38 pathway. These results suggest that emodin may provide an effective therapeutic agent for fibrosis that functions via specific TGF-ß signaling pathways.

Effects of the isothiocyanate sulforaphane on TGF-ß1-induced rat cardiac fibroblast activation and extracellular matrix interactions.

An important step in many pathological conditions, particularly tissue and organ fibrosis, is the conversion of relatively quiescent cells into active myofibroblasts. These are highly specialized cells that participate in normal wound healing but also contribute to pathogenesis. These cells possess characteristics of smooth muscle cells and fibroblasts, have enhanced synthetic activity secreting abundant extracellular matrix components, cytokines, and growth factors, and are capable of generating contractile force. As such, these cells have become potential therapeutic targets in a number of disease settings. Transforming growth factor ß (TGF-ß) is a potent stimulus of fibrosis and myofibroblast formation and likewise is an important therapeutic target in several disease conditions. The plant-derived isothiocyanate sulforaphane has been shown to have protective effects in several pathological models including diabetic cardiomyopathy, carcinogenesis, and fibrosis. These studies suggest that sulforaphane may be an attractive preventive agent against disease progression, particularly in conditions involving alterations of the extracellular matrix and activation of myofibroblasts. However, few studies have evaluated the effects of sulforaphane on cardiac fibroblast activation and their interactions with the extracellular matrix. The present studies were carried out to determine the potential effects of sulforaphane on the conversion of quiescent cardiac fibroblasts to an activated myofibroblast phenotype and associated alterations in signaling, expression of extracellular matrix receptors, and cellular physiology following stimulation with TGF-ß1. These studies demonstrate that sulforaphane attenuates TGF-ß1-induced myofibroblast formation and contractile activity. Sulforaphane also reduces expression of collagen-binding integrins and inhibits canonical and noncanonical TGF-ß signaling pathways.

Meltblown Polylactic Acid Nanowebs as a Tissue Engineering Scaffold.

Polylactic acid (PLA) nanofiber nonwovens have recently come under more vigorous investigation for their use as tissue engineering scaffolds owing to its ability to mimic the physical properties of naturally occurring human extracellular matrix in a variety of host tissues. Currently, the majority of available research on PLA nanowebs has focused on their creation through electrospinning. The goal of this study was to evaluate meltblown nonwoven webs made of nanodiameter PLA fibers for their application as a tissue engineering scaffold. Meltblown PLA fabrics were produced with a variety of different crystallinities, tensile moduli, and pore diameters. One fabric with mechanical properties similar to human dermis was selected as a scaffold to study attachment, proliferation, and migration of human dermal fibroblasts over 1, 3, 7, and 14 days without the use of additional cell adhesion molecules. The 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide assay showed good proliferation from day 1 to 3 (P = 0.026) and up to 7 days of culture (P = 0.005) but without increase from day 7 to 14. Electron microscopy demonstrated adequate cellular attachment and surface migration at 1, 3, 7, and 14 days. Finally, confocal microscopy was used to investigate cellular penetration into the scaffolds. The investigation found that cells were able to penetrate fully through the thickness of the scaffold. The successes of this initial experiment are promising and confirm that meltblown nanofiber nonwovens are a viable avenue for tissue engineering scaffolds. Hopefully, these conclusions will open the door for others to pursue research in this exciting field.

Acute myotube protein synthesis regulation by IL-6-related cytokines.

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis.

Potential of Plant-Derived Compounds in Preventing and Reversing Organ Fibrosis and the Underlying Mechanisms.

Increased production of extracellular matrix is a necessary response to tissue damage and stress. In a normal healing process, the increase in extracellular matrix is transient. In some instances; however, the increase in extracellular matrix can persist as fibrosis, leading to deleterious alterations in organ structure, biomechanical properties, and function. Indeed, fibrosis is now appreciated to be an important cause of mortality and morbidity. Extensive research has illustrated that fibrosis can be slowed, arrested or even reversed; however, few drugs have been approved specifically for anti-fibrotic treatment. This is in part due to the complex pathways responsible for fibrogenesis and the undesirable side effects of drugs targeting these pathways. Natural products have been utilized for thousands of years as a major component of traditional medicine and currently account for almost one-third of drugs used clinically worldwide. A variety of plant-derived compounds have been demonstrated to have preventative or even reversal effects on fibrosis. This review will discuss the effects and the underlying mechanisms of some of the major plant-derived compounds that have been identified to impact fibrosis.

Activation of cardiac fibroblasts by ethanol is blocked by TGF-ß inhibition.

BACKGROUND: Alcohol abuse is the second leading cause of dilated cardiomyopathy, a disorder specifically referred to as alcoholic cardiomyopathy (ACM). Rodent and human studies have revealed cardiac fibrosis to be a consequence of ACM, and prior studies by this laboratory have associated this occurrence with elevated transforming growth factor-beta (TGF-ß) and activated fibroblasts (myofibroblasts). To date, there have been no other studies to investigate the direct effect of alcohol on the cardiac fibroblast. METHODS: Primary rat cardiac fibroblasts were cultured in the presence of ethanol (EtOH) and assayed for fibroblast activation by collagen gel contraction, alpha-smooth muscle actin (α-SMA) expression, migration, proliferation, apoptosis, collagen I and III, and TGF-ß expression. The TGF-ß receptor type 1 inhibitor compound SB 431542 and a soluble recombinant TGF-ßII receptor (RbII) were used to assess the role of TGF-ß in the response of cardiac fibroblasts to EtOH. RESULTS: Treatment for cardiac fibroblasts with EtOH at concentrations of 100 mg/dl or higher resulted in fibroblast activation and fibrogenic activity after 24 hours including an increase in contraction, α-SMA expression, migration, and expression of collagen I and TGF-ß. No changes in fibroblast proliferation or apoptosis were observed. Inhibition of TGF-ß by SB 431542 and RbII attenuated the EtOH-induced fibroblast activation. CONCLUSIONS: EtOH treatment directly promotes cardiac fibroblast activation by stimulating TGF-ß release from fibroblasts. Inhibiting the action of TGF-ß decreases the fibrogenic effect induced by EtOH treatment. The results of this study support TGF-ß to be an important component in cardiac fibrosis induced by exposure to EtOH.

The SARS-CoV-2 spike protein induces long-term transcriptional perturbations of mitochondrial metabolic genes, causes cardiac fibrosis, and reduces myocardial contractile in obese mice.

BACKGROUND: As the pandemic evolves, post-acute sequelae of CoV-2 (PASC) including cardiovascular manifestations have emerged as a new health threat. This study aims to study whether the Spike protein plus obesity can exacerbate PASC-related cardiomyopathy. METHODS: A Spike protein-pseudotyped (Spp) virus with the proper surface tropism of SARS-CoV-2 was developed for viral entry assay in vitro and administration into high fat diet (HFD)-fed mice. The systemic viral loads and cardiac transcriptomes were analyzed at 2 and 24 h, 3, 6, and 24 weeks post introducing (wpi) Spp using RNA-seq or real time RT-PCR. Echocardiography was used to monitor cardiac functions. RESULTS: Low-density lipoprotein cholesterol enhanced viral uptake in endothelial cells, macrophages, and cardiomyocyte-like H9C2 cells. Selective cardiac and adipose viral depositions were observed in HFD mice but not in normal-chow-fed mice. The cardiac transcriptional signatures in HFD mice at 3, 6, and 24 wpi showed systemic suppression of mitochondria respiratory chain genes including ATP synthases and nicotinamide adenine dinucleotide:ubiquinone oxidoreductase gene members, upregulation of stress pathway-related crucial factors such as nuclear factor-erythroid 2-related factor 1 and signal transducer and activator of transcription 5A, and increases in expression of glucose metabolism-associated genes. As compared with the age-matched HFD control mice, cardiac ejection fraction and fractional shortening were significantly decreased, while left ventricular end-systolic diameter and volume were significantly elevated, and cardiac fibrosis was increased in HFD mice at 24 wpi. CONCLUSION: Our data demonstrated that the Spike protein could induce long-term transcriptional suppression of mitochondria metabolic genes and cause cardiac fibrosis and myocardial contractile impairment in obese mice, providing mechanistic insights to PASC-related cardiomyopathy.

The SARS-CoV-2 Spike protein induces long-term transcriptional perturbations of mitochondrial metabolic genes, causes cardiac fibrosis, and reduces myocardial contractile in obese mice.

Background: As the pandemic evolves, post-acute sequelae of CoV-2 (PACS) including cardiovascular manifestations have emerged as a new health threat. This study aims to study whether the Spike protein plus obesity can exacerbate PACS-related cardiomyopathy. Methods: A Spike protein-pseudotyped (Spp) virus with the proper surface tropism of SARS-CoV-2 was developed for viral entry assay in vitro and administration into high fat diet (HFD)-fed mice. The systemic viral loads and cardiac transcriptomes were analyzed at 2 and 24 hrs, 3, 6, and 24 weeks post introducing (wpi) Spp using RNA-seq or real time RT-PCR. Echocardiography was used to monitor cardiac functions. Results: Low-density lipoprotein cholesterol enhanced viral uptake in endothelial cells, macrophages, and cardiomyocyte-like H9C2 cells. Selective cardiac and adipose viral depositions were observed in HFD mice but not in normal-chow-fed mice. The cardiac transcriptional signatures in HFD mice at 3, 6, and 24 wpi showed systemic suppression of mitochondria respiratory chain genes including ATP synthases and nicotinamide adenine dinucleotide:ubiquinone oxidoreductase gene members, upregulation of stress pathway-related crucial factors such as nuclear factor-erythroid 2-related factor 1 and signal transducer and activator of transcription 5A, and increases in expression of glucose metabolism-associated genes. As compared with the age-matched HFD control mice, cardiac ejection fraction and fractional shortening were significantly decreased, while left ventricular end-systolic diameter and volume were significantly elevated, and cardiac fibrosis was increased in HFD mice at 24 wpi. Conclusion: Our data demonstrated that the Spike protein could induce long-term transcriptional suppression of mitochondria metabolic genes and cause cardiac fibrosis and myocardial contractile impairment, providing mechanistic insights to PACS-related cardiomyopathy.

Development of a bioactive tunable hyaluronic-protein bioconjugate hydrogel for tissue regenerative applications.

Every year, there are approximately 500 000 peripheral nerve injury (PNI) procedures due to trauma in the US alone. Autologous and acellular nerve grafts are among current clinical repair options; however, they are limited largely by the high costs associated with donor nerve tissue harvesting and implant processing, respectively. Therefore, there is a clinical need for an off-the-shelf nerve graft that can recapitulate the native microenvironment of the nerve. In our previous work, we created a hydrogel scaffold that incorporates mechanical and biological cues that mimic the peripheral nerve microenvironment using chemically modified hyaluronic acid (HA). However, with our previous work, the degradation profile and cell adhesivity was not ideal for tissue regeneration, in particular, peripheral nerve regeneration. To improve our previous hydrogel, HA was conjugated with fibrinogen using Michael-addition to assist in cell adhesion and hydrogel degradability. The addition of the fibrinogen linker was found to contribute to faster scaffold degradation via active enzymatic breakdown, compared to HA alone. Additionally, cell count and metabolic activity was significantly higher on HA conjugated fibrinogen compared previous hydrogel formulations. This manuscript discusses the various techniques deployed to characterize our new modified HA fibrinogen chemistry physically, mechanically, and biologically. This work addresses the aforementioned concerns by incorporating controllable degradability and increased cell adhesivity while maintaining incorporation of hyaluronic acid, paving the pathway for use in a variety of applications as a multi-purpose tissue engineering platform.

Effects of interleukin-33 on cardiac fibroblast gene expression and activity.

Interleukin-33 (IL-33) is a recently described member of the interleukin-1 (IL-1) family. It is produced by diverse cell types in response to a variety of stresses including hemorrhage and increased mechanical load. Though only relatively recently discovered, IL-33 has been shown to participate in several pathological processes including promoting type 2 T helper cell-associated autoimmune diseases. In contrast, IL-33 has been also found to have protective effects in cardiovascular diseases. Recent studies have illustrated that IL-33 attenuates cardiac fibrosis induced by increased cardiovascular load in mice (transaortic constriction). Since cardiac fibrosis is largely dependent on increased production of extracellular matrix by cardiac fibroblasts, we hypothesized that IL-33 directly inhibits pro-fibrotic activities of these cells. Experiments have been carried out with isolated rat cardiac fibroblasts to evaluate the effects of IL-33 on the modulation of cardiac fibroblast gene expression and function to test this hypothesis. The expression of the IL-33 receptor, interleukin-1 receptor-like 1 (ST2), was detected at the mRNA and protein levels in isolated adult rat cardiac fibroblasts. Subsequently, the effects of IL-33 treatment (0-100 ng/ml) on the expression of extracellular matrix proteins and pro-inflammatory cytokines/chemokines were examined as well as the effects on rat cardiac fibroblast activities including proliferation, collagen gel contraction and migration. While IL-33 did not directly inhibit collagen I and collagen III production, it yielded a dose-dependent increase in the expression of interleukin-6 and monocyte chemotactic protein-1. Treatment of rat cardiac fibroblasts with IL-33 also impaired the migratory activity of these cells. Further experiments illustrated that IL-33 rapidly activated multiple signaling pathways including extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in a dose-dependent manner. Experiments were carried out with pharmacological inhibitors to determine the role of specific signaling pathways in the response of fibroblasts to IL-33. These experiments illustrated that the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases are critical to the increased production of interleukin-6 and monocyte chemotactic protein-1 in response to IL-33. These studies suggest that IL-33 has an important role in the modulation of fibroblast function and gene expression. Surprisingly, IL-33 had no effect on the expression of genes encoding extracellular matrix components or on proliferation, markers typical of fibrosis. The major effects of IL-33 detected in these studies included inhibition of cell migration and activation of cytokine/chemokine expression. The previously reported inhibition of cardiac fibrosis may include more complicated mechanisms that involve other cardiac cell types. Future studies aimed at determining the effects of IL-33 on other cardiac cell types are warranted.

Alterations in cardiac structure and function in a murine model of chronic alcohol consumption.

Male, wild-type, FVB strain mice were fed a nutritionally complete liquid diet supplemented with 4% ethanol v/v over a time course of 1, 2, 4, 8, 12, and 14 weeks. Controls were offered an isocaloric liquid equivalent and pair fed with their ethanol counterparts. Changes in cardiac physiology were assessed at respective time points via echocardiography. Additionally, the use of histological techniques, mRNA analysis, apoptosis determination, and immunohistochemistry were employed to determine the functional and structural changes on the heart. Echocardiograph analysis revealed a compensatory phase that occurred early in the time course (1-8 weeks) and decompensation reverting toward heart failure at weeks 12 and 14. Throughout the study, an increase in cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis, TGF-ß, and the presence of α-SMA-positive cells were determined. A compensatory period in mice treated with ethanol occurred early followed by a transition to a dilated phenotype over time. A number of factors may be involved in this process including the activation of myofibroblasts and their fibrotic activities that is correlated with the presence of transforming growth factor beta.

Diabetes-induced alterations in the extracellular matrix and their impact on myocardial function.

Diabetes is an increasing public health problem that is expected to escalate in the future due to the growing incidence of obesity in the western world. While this disease is well known for its devastating effects on the kidneys and vascular system, diabetic individuals can develop cardiac dysfunction, termed diabetic cardiomyopathy, in the absence of other cardiovascular risk factors such as hypertension or atherosclerosis. While much effort has gone into understanding the effects of elevated glucose or altered insulin sensitivity on cellular components within the heart, significant changes in the cardiac extracellular matrix (ECM) have also been noted. In this review article we highlight what is currently known regarding the effects diabetes has on both the expression and chemical modification of proteins within the ECM and how the fibrotic response often observed as a consequence of this disease can contribute to reduced cardiac function.

The Spike Protein of SARS-CoV-2 Impairs Lipid Metabolism and Increases Susceptibility to Lipotoxicity: Implication for a Role of Nrf2.

Coronavirus disease 2019 (COVID-19) patients show lipid metabolic alterations, but the mechanism remains unknown. In this study, we aimed to investigate whether the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs lipid metabolism in host cells. We generated a Spike cell line in HEK293 using the pcDNA vector carrying the Spike gene expression cassette. A control cell line was generated using the empty pcDNA vector. Gene expression profiles related to lipid metabolic, autophagic, and ferroptotic pathways were investigated. Palmitic acid (PA)-overload was used to assess lipotoxicity-induced necrosis. As compared with controls, the Spike cells showed a significant increase in lipid depositions in cell membranes as well as dysregulation of expression of a panel of molecules involving lipid metabolism, autophagy, and ferroptosis. The Spike cells showed an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a multifunctional transcriptional factor, in response to PA. Furthermore, the Spike cells exhibited increased necrosis in response to PA-induced lipotoxicity compared to control cells in a time- and dose-dependent manner via ferroptosis, which could be attenuated by the Nrf2 inhibitor trigonelline. We conclude that the Spike protein impairs lipid metabolic and autophagic pathways in host cells, leading to increased susceptibility to lipotoxicity via ferroptosis which can be suppressed by a Nrf2 inhibitor. This data also suggests a central role of Nrf2 in Spike-induced lipid metabolic impairments.

Small-diameter artery decellularization: Effects of anionic detergent concentration and treatment duration on porcine internal thoracic arteries.

Engineered replacement materials have tremendous potential for vascular applications where over 400,000 damaged and diseased blood vessels are replaced annually in the United States alone. Unlike large diameter blood vessels, which are effectively replaced by synthetic materials, prosthetic small-diameter vessels are prone to early failure, restenosis, and reintervention surgery. We investigated the differential response of varying 0%-6% sodium dodecyl sulfate and sodium deoxycholate anionic detergent concentrations after 24 and 72 h in the presence of DNase using biochemical, histological, and biaxial mechanical analyses to optimize the decellularization process for xenogeneic vascular tissue sources, specifically the porcine internal thoracic artery (ITA). Detergent concentrations greater than 1% were successful at removing cytoplasmic and cell surface proteins but not DNA content after 24 h. A progressive increase in porosity and decrease in glycosaminoglycan (GAG) content was observed with detergent concentration. Augmented porosity was likely due to the removal of both cells and GAGs and could influence recellularization strategies. The treatment duration on the other hand, significantly improved decellularization by reducing DNA content to trace amounts after 72 h. Prolonged treatment times reduced laminin content and influenced the vessel's mechanical behavior in terms of altered circumferential stress and stretch while further increasing porosity. Collectively, DNase with 1% detergent for 72 h provided an effective and efficient decellularization strategy to be employed in the preparation of porcine ITAs as bypass graft scaffolding materials with minor biomechanical and histological penalties.

Effects of interleukin-18 on cardiac fibroblast function and gene expression.

Fibroblasts are the primary cell type responsible for synthesis and remodeling of the extracellular matrix in the heart. A number of factors including growth factors, hormones and mechanical forces have been identified that modulate the production of extracellular matrix by cardiac fibroblasts. Inflammatory mediators including pro-inflammatory cytokines and chemokines also impact fibrosis of the heart. Recent studies have illustrated that interleukin-18 promotes a pro-fibrotic response in cardiac fibroblasts; however the effects of this cytokine on other aspects of fibroblast function have not been examined. While fibroblasts have long been known for their role in production and remodeling of the extracellular matrix, other functions of these cells are only now beginning to be appreciated. We hypothesize that exposure to interleukin-18 will stimulate other aspects of fibroblast behavior important in myocardial remodeling including proliferation, migration and collagen reorganization. Fibroblasts were isolated from adult male rat hearts and bioassays performed to determine the effects of interleukin-18 on fibroblast function. Treatment of fibroblasts with interleukin-18 (1-100ng/ml) resulted in increased production of extracellular matrix components and remodeling or contraction of three-dimensional collagen scaffolds by these cells. Furthermore, exposure to interleukin-18 stimulated fibroblast migration and proliferation. Treatment of heart fibroblasts with interleukin-18 resulted in the rapid activation of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3-kinase) pathways. Studies with pharmacological inhibitors illustrated that activation of these pathways is critical to interleukin-18 mediated alterations in fibroblast function. These studies illustrate that interleukin-18 plays a role in modulation of cardiac fibroblast function and may be an important component of the inflammation-fibrosis cascade during pathological myocardial remodeling.

Longitudinal histomechanical heterogeneity of the internal thoracic artery.

The internal thoracic artery (ITA) is the principal choice for coronary artery bypass grafting (CABG) due to its mechanical compatibility, histological composition, anti-thrombogenic lumen, and single anastomotic junction. Originating at the subclavian artery, traversing the thoracic cavity, and terminating at the superior epigastric and musculophrenic bifurcation, bilateral ITAs follow a protracted circuitous pathway. The physiological hemodynamics, anatomical configuration, and perivascular changes that occur throughout this length influence the tissue's microstructure and gross mechanical properties. Since histomechanics play a major role in premature graft failure we used inflation-extension testing to quantify the regional material and biaxial mechanical properties at four distinct locations along the left (L) and right (R) ITA and fit the results to a structurally-motivated constitutive model. Our comparative analysis of 44 vessel segments revealed a significant increase in the amount of collagen but not smooth muscle and a significant decrease in elastin and elastic lamellae present with distance from the heart. A subsequent decrease in the total deformation energy and isotropic contribution to the strain energy was present in the LITA but not RITA. Circumferential stress and compliance generally decreased along the length of the LITA while axial stress increased in the RITA. When comparing RITAs to LITAs, some morphological and histological differences were found in proximal sections while distal sections revealed differences predominantly in compliance and axial stress. Overall, this information can be used to better guide graft selection, graft preparation, and xenograft-based tissue-engineering strategies for CABG.

Roles of Exosomes in Cardiac Fibroblast Activation and Fibrosis.

Alterations in the accumulation and composition of the extracellular matrix are part of the normal tissue repair process. During fibrosis, this process becomes dysregulated and excessive extracellular matrix alters the biomechanical properties and function of tissues involved. Historically fibrosis was thought to be progressive and irreversible; however, studies suggest that fibrosis is a dynamic process whose progression can be stopped and even reversed. This realization has led to an enhanced pursuit of therapeutic agents targeting fibrosis and extracellular matrix-producing cells. In many organs, fibroblasts are the primary cells that produce the extracellular matrix. In response to diverse mechanical and biochemical stimuli, these cells are activated or transdifferentiate into specialized cells termed myofibroblasts that have an enhanced capacity to produce extracellular matrix. It is clear that interactions between diverse cells of the heart are able to modulate fibroblast activation and fibrosis. Exosomes are a form of extracellular vesicle that play an important role in intercellular communication via the cargo that they deliver to target cells. While relatively recently discovered, exosomes have been demonstrated to play important positive and negative roles in the regulation of fibroblast activation and tissue fibrosis. These roles as well as efforts to engineer exosomes as therapeutic tools will be discussed.

Temporal alterations in cardiac fibroblast function following induction of pressure overload.

Increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis. While numerous studies have focused on the mechanisms of myocyte hypertrophy, comparatively little is known regarding the response of the interstitial fibroblasts to increased cardiovascular load. Fibroblasts are the most numerous cell type in the mammalian myocardium and have long been recognized as producing the majority of the myocardial extracellular matrix. It is only now becoming appreciated that other aspects of fibroblast behavior are important to overall cardiac function. The present studies were performed to examine the temporal alterations in fibroblast activity in response to increased cardiovascular load. Rat myocardial fibroblasts were isolated at specific time-points (3, 7, 14, and 28 days) after induction of pressure overload by abdominal aortic constriction. Bioassays were performed to measure specific parameters of fibroblast function including remodeling and contraction of 3-dimensional collagen gels, migration, and proliferation. In addition, the expression of extracellular matrix receptors of the integrin family was examined. Myocardial hypertrophy and fibrosis were evident within 7 days after constriction of the abdominal aorta. Collagen gel contraction, migration, and proliferation were enhanced in fibroblasts from pressure-overloaded animals compared to fibroblasts from sham animals. Differences in fibroblast function and protein expression were evident within 7 days of aortic constriction, concurrent with the onset of hypertrophy and fibrosis of the intact myocardium. These data provide further support for the idea that rapid and dynamic changes in fibroblast phenotype accompany and contribute to the progression of cardiovascular disease.

Spike Protein of SARS-CoV-2 Activates Macrophages and Contributes to Induction of Acute Lung Inflammations in Mice.

Background: Coronavirus disease 2019 (COVID-19) patients exhibit multiple organ malfunctions with a primary manifestation of acute and diffuse lung injuries. The Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to mediate viral entry into host cells; however, whether it can be cellularly pathogenic and contribute to pulmonary hyper-inflammations in COVID-19 is not well known. Methods and Findings: In this study, we developed a Spike protein-pseudotyped (Spp) lentivirus with the proper tropism of SARS-CoV-2 Spike protein on the surface and tracked down the fate of Spp in wild type C57BL/6J mice receiving intravenous injection of the virus. A lentivirus with vesicular stomatitis virus glycoprotein (VSV-G) was used as the control. Two hours post-infection (hpi), Spp showed more than 27-75 times more viral burden in the lungs than other organs; it also exhibited about 3-5 times more viral burden than VSV-G lentivirus in the lungs, liver, kidney and spleen. Acute pneumonia was evident in animals 24 hpi. Spp lentivirus was mainly found in LDLR+ macrophages and pneumocytes in the lungs, but not in MARC1+ macrophages. IL6, IL10, CD80 and PPAR-γ were quickly upregulated in response to infection of Spp lentivirus in the lungs in vivo as well as in macrophage-like RAW264.7 cells in vitro. We further confirmed that forced expression of the Spike protein in RAW264.7 cells could significantly increase the mRNA levels of the same panel of inflammatory factors. Conclusions: Our results demonstrate that the Spike protein of SARS-CoV-2 alone can induce cellular pathology, e.g. activating macrophages and contributing to induction of acute inflammatory responses.