SPADE: spatial deconvolution for domain specific cell-type estimation.

Understanding gene expression in different cell types within their spatial context is a key goal in genomics research. SPADE (SPAtial DEconvolution), our proposed method, addresses this by integrating spatial patterns into the analysis of cell type composition. This approach uses a combination of single-cell RNA sequencing, spatial transcriptomics, and histological data to accurately estimate the proportions of cell types in various locations. Our analyses of synthetic data have demonstrated SPADE's capability to discern cell type-specific spatial patterns effectively. When applied to real-life datasets, SPADE provides insights into cellular dynamics and the composition of tumor tissues. This enhances our comprehension of complex biological systems and aids in exploring cellular diversity. SPADE represents a significant advancement in deciphering spatial gene expression patterns, offering a powerful tool for the detailed investigation of cell types in spatial transcriptomics.

Urocortin2 attenuates diabetic coronary microvascular dysfunction by regulating macrophage extracellular vesicles.

Diabetic patients develop coronary microvascular dysfunction (CMD) and exhibit high mortality of coronary artery disease. Methylglyoxal (MGO) largely accumulates in the circulation due to diabetes. We addressed whether macrophages exposed to MGO exhibited damaging effect on the coronary artery and whether urocortin2 (UCN2) serve as protecting factors against such diabetes-associated complication. Type 2 diabetes was induced by high-fat diet and a single low-dose streptozotocin in mice. Small extracellular vesicles (sEV) derived from MGO-treated macrophages (MGO-sEV) were used to produce diabetes-like CMD. UCN2 was examined for a protective role against CMD. The involvement of arginase1 and IL-33 was tested by pharmacological inhibitor and IL-33-/- mice. MGO-sEV was capable of causing coronary artery endothelial dysfunction similar to that by diabetes. Immunocytochemistry studies of diabetic coronary arteries supported the transfer of arginase1 from macrophages to endothelial cells. Mechanism studies revealed arginase1 contributed to the impaired endothelium-dependent relaxation of coronary arteries in diabetic and MGO-sEV-treated mice. UCN2 significantly improved coronary artery endothelial function, and prevented MGO elevation in diabetic mice or enrichment of arginase1 in MGO-sEV. Diabetes caused a reduction of IL-33, which was also reversed by UCN2. IL-33-/- mice showed impaired endothelium-dependent relaxation of coronary arteries, which can be mitigated by arginase1 inhibition but can't be improved by UCN2 anymore, indicating the importance of restoring IL-33 for the protection against diabetic CMD by UCN2. Our data suggest that MGO-sEV induces CMD via shuttling arginase1 to coronary arteries. UCN2 is able to protect against diabetic CMD via modulating MGO-altered macrophage sEV cargoes.

Semi-reference based cell type deconvolution with application to human metastatic cancers.

Bulk RNA-seq experiments, commonly used to discern gene expression changes across conditions, often neglect critical cell type-specific information due to their focus on average transcript abundance. Recognizing cell type contribution is crucial to understanding phenotype and disease variations. The advent of single-cell RNA sequencing has allowed detailed examination of cellular heterogeneity; however, the cost and analytic caveat prohibits such sequencing for a large number of samples. We introduce a novel deconvolution approach, SECRET, that employs cell type-specific gene expression profiles from single-cell RNA-seq to accurately estimate cell type proportions from bulk RNA-seq data. Notably, SECRET can adapt to scenarios where the cell type present in the bulk data is unrepresented in the reference, thereby offering increased flexibility in reference selection. SECRET has demonstrated superior accuracy compared to existing methods using synthetic data and has identified unknown tissue-specific cell types in real human metastatic cancers. Its versatility makes it broadly applicable across various human cancer studies.

Del Nido cardioplegia or potassium induces Nrf2 and protects cardiomyocytes against oxidative stress.

Open heart surgery is often an unavoidable procedure for the treatment of coronary artery disease. The procedure-associated reperfusion injury affects postoperative cardiac performance and long-term outcomes. We addressed here whether cardioplegia essential for cardiopulmonary bypass surgery activates Nrf2, a transcription factor regulating the expression of antioxidant and detoxification genes. With commonly used cardioplegic solutions, high K+, low K+, Del Nido (DN), histidine-tryptophan-ketoglutarate (HTK), and Celsior (CS), we found that DN caused a significant increase of Nrf2 protein in AC16 human cardiomyocytes. Tracing the ingredients in DN led to the discovery of KCl at the concentration of 20-60 mM capable of significant Nrf2 protein induction. The antioxidant response element (ARE) luciferase reporter assays confirmed Nrf2 activation by DN or KCl. Transcriptomic profiling using RNA-seq revealed that oxidation-reduction as a main gene ontology group affected by KCl. KCl indeed elevated the expression of classical Nrf2 downstream targets, including TXNRD1, AKR1C, AKR1B1, SRXN1, and G6PD. DN or KCl-induced Nrf2 elevation is Ca2+ concentration dependent. We found that KCl decreased Nrf2 protein ubiquitination and extended the half-life of Nrf2 from 17.8 to 25.1 mins. Knocking out Keap1 blocked Nrf2 induction by K+. Nrf2 induction by DN or KCl correlates with the protection against reactive oxygen species generation or loss of viability by H2O2 treatment. Our data support that high K+ concentration in DN cardioplegic solution can induce Nrf2 protein and protect cardiomyocytes against oxidative damage.NEW & NOTEWORTHY Open heart surgery is often an unavoidable procedure for the treatment of coronary artery disease. The procedure-associated reperfusion injury affects postoperative cardiac performance and long-term outcomes. We report here that Del Nido cardioplegic solution or potassium is an effective inducer of Nrf2 transcription factor, which controls the antioxidant and detoxification response. This indicates that Del Nido solution is not only essential for open heart surgery but also exhibits cardiac protective activity.

Hyperkalemic or Low Potassium Cardioplegia Protects against Reduction of Energy Metabolism by Oxidative Stress.

Open-heart surgery is often an unavoidable option for the treatment of cardiovascular disease and prevention of cardiomyopathy. Cardiopulmonary bypass surgery requires manipulating cardiac contractile function via the perfusion of a cardioplegic solution. Procedure-associated ischemia and reperfusion (I/R) injury, a major source of oxidative stress, affects postoperative cardiac performance and long-term outcomes. Using large-scale liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics, we addressed whether cardioplegic solutions affect the baseline cellular metabolism and prevent metabolic reprogramming by oxidative stress. AC16 cardiomyocytes in culture were treated with commonly used cardioplegic solutions, High K+ (HK), Low K+ (LK), Del Nido (DN), histidine-tryptophan-ketoglutarate (HTK), or Celsior (CS). The overall metabolic profile shown by the principal component analysis (PCA) and heatmap revealed that HK or LK had a minimal impact on the baseline 78 metabolites, whereas HTK or CS significantly repressed the levels of multiple amino acids and sugars. H2O2-induced sublethal mild oxidative stress causes decreases in NAD, nicotinamide, or acetylcarnitine, but increases in glucose derivatives, including glucose 6-P, glucose 1-P, fructose, mannose, and mannose 6-P. Additional increases include metabolites of the pentose phosphate pathway, D-ribose-5-P, L-arabitol, adonitol, and xylitol. Pretreatment with HK or LK cardioplegic solution prevented most metabolic changes and increases of reactive oxygen species (ROS) elicited by H2O2. Our data indicate that HK and LK cardioplegic solutions preserve baseline metabolism and protect against metabolic reprogramming by oxidative stress.

Fresh Medium or L-Cystine as an Effective Nrf2 Inducer for Cytoprotection in Cell Culture.

The Nrf2 gene encodes a transcription factor best known for regulating the expression of antioxidant and detoxification genes. A long list of small molecules has been reported to induce Nrf2 protein via Keap1 oxidation or alkylation. Many of these Nrf2 inducers exhibit off-target or toxic effects due to their nature as electrophiles. In searching for non-toxic Nrf2 inducers, we found that a culture medium change to fresh DMEM is capable of inducing Nrf2 protein in HeLa, HEK293, AC16 and MCF7 cells. Testing the components of DMEM led to the discovery of L-Cystine as an effective Nrf2 inducer. L-Cystine induces a dose-dependent increase of Nrf2 protein, from 0.1 to 1.6 mM. RNA-seq analyses and RT-PCR revealed an induction of multiple Nrf2 downstream genes, including NQO1, HMOX1, GCLC, GCLM, SRXN1, TXNRD1, AKR1C and OSGIN1 by 0.8 mM L-Cystine. The induction of Nrf2 protein was dependent on L-Cystine entering cells via the cystine/glutamate antiporter and the presence of Keap1. The half-life of Nrf2 protein increased from 19.4 min to 30.9 min with 0.8 mM L-Cystine treatment. L-Cystine was capable of eliciting cytoprotection by reducing ROS generation and protecting against oxidant- or doxorubicin-induced apoptosis. As an amino acid derivative, L-Cystine is considered a non-toxic Nrf2 inducer that exhibits the potential for protection against oxidative stress and tissue injury.

Metabolomics of oxidative stress: Nrf2 independent depletion of NAD or increases of sugar alcohols.

Nrf2 encodes a transcription factor best known for regulating the expression of antioxidant and detoxification genes. Recent evidence suggested that Nrf2 mediates metabolic reprogramming in cancer cells. However, the role of Nrf2 in the biochemical metabolism of cardiac cells has not been studied. Using LC-MS/MS-based metabolomics, we addressed whether knocking out the Nrf2 gene in AC16 human cardiomyocytes affects metabolic reprogramming by oxidative stress. Profiling the basal level metabolites showed an elevated pentose phosphate pathway and increased levels of sugar alcohols, sorbitol, L-arabitol, xylitol and xylonic acid, in Nrf2 KO cells. With sublethal levels of oxidative stress, depletion of NAD, an increase of GDP and elevation of sugar alcohols, sorbitol and dulcitol, were detected in parent wild type (WT) cells. Knocking out Nrf2 did not affect these changes. Biochemical assays confirmed depletion of NAD in WT and Nrf2 KO cells due to H2O2 treatment. These data support that although Nrf2 deficiency caused baseline activation of the pentose phosphate pathway and sugar alcohol synthesis, a brief exposure to none-lethal doses of H2O2 caused NAD depletion in an Nrf2 independent manner. Loss of NAD may contribute to oxidative stress associated cell degeneration as observed with aging, diabetes and heart failure.

Nrf2 for protection against oxidant generation and mitochondrial damage in cardiac injury.

Myocardial infarction is the most common form of acute coronary syndrome. Blockage of a coronary artery due to blood clotting leads to ischemia and subsequent cell death in the form of necrosis, apoptosis, necroptosis and ferroptosis. Revascularization by coronary artery bypass graft surgery or non-surgical percutaneous coronary intervention combined with pharmacotherapy is effective in relieving symptoms and decreasing mortality. However, reactive oxygen species (ROS) are generated from damaged mitochondria, NADPH oxidases, xanthine oxidase, and inflammation. Impairment of mitochondria is shown as decreased metabolic activity, increased ROS production, membrane permeability transition, and release of mitochondrial proteins into the cytoplasm. Oxidative stress activates Nrf2 transcription factor, which in turn mediates the expression of mitofusin 2 (Mfn 2) and proteasomal genes. Increased expression of Mfn2 and inhibition of mitochondrial fission due to decreased Drp1 protein by proteasomal degradation contribute to mitochondrial hyperfusion. Damaged mitochondria can be removed by mitophagy via Parkin or p62 mediated ubiquitination. Mitochondrial biogenesis compensates for the loss of mitochondria, but requires mitochondrial DNA replication and initiation of transcription or translation of mitochondrial genes. Experimental evidence supports a role of Nrf2 in mitophagy, via up-regulation of PINK1 or p62 gene expression; and in mitochondrial biogenesis, by influencing the expression of PGC-1α, NResF1, NResF2, TFAM and mitochondrial genes. Oxidative stress causes Nrf2 activation via Keap1 dissociation, de novo protein translation, and nuclear translocation related to inactivation of GSK3ß. The mechanism of Keap 1 mediated Nrf2 activation has been hijacked for Nrf2 activation by small molecules derived from natural products, some of which have been shown capable of mitochondrial protection. Multiple lines of evidence support the importance of Nrf2 in protecting mitochondria and preserving or renewing energy metabolism following tissue injury.

Nrf2 at the heart of oxidative stress and cardiac protection.

The NFE2L2 gene encodes the transcription factor Nrf2 best known for regulating the expression of antioxidant and detoxification genes. Gene knockout approaches have demonstrated its universal cytoprotective features. While Nrf2 has been the topic of intensive research in cancer biology since its discovery in 1994, understanding the role of Nrf2 in cardiovascular disease has just begun. The literature concerning Nrf2 in experimental models of atherosclerosis, ischemia, reperfusion, cardiac hypertrophy, heart failure, and diabetes supports its cardiac protective character. In addition to antioxidant and detoxification genes, Nrf2 has been found to regulate genes participating in cell signaling, transcription, anabolic metabolism, autophagy, cell proliferation, extracellular matrix remodeling, and organ development, suggesting that Nrf2 governs damage resistance as well as wound repair and tissue remodeling. A long list of small molecules, most derived from natural products, have been characterized as Nrf2 inducers. These compounds disrupt Keap1-mediated Nrf2 ubquitination, thereby prohibiting proteasomal degradation and allowing Nrf2 protein to accumulate and translocate to the nucleus, where Nrf2 interacts with sMaf to bind to ARE in the promoter of genes. Recently alternative mechanisms driving Nrf2 protein increase have been revealed, including removal of Keap1 by autophagy due to p62/SQSTM1 binding, inhibition of ßTrCP or Synoviolin/Hrd1-mediated ubiquitination of Nrf2, and de novo Nrf2 protein translation. We review here a large volume of literature reporting historical and recent discoveries about the function and regulation of Nrf2 gene. Multiple lines of evidence presented here support the potential of dialing up the Nrf2 pathway for cardiac protection in the clinic.

Differential Regulation of Bcl-xL Gene Expression by Corticosterone, Progesterone, and Retinoic Acid.

Corticosterone (CT), progesterone (PG), and retinoic acid (RA) are capable of inhibiting Doxorubicin (Dox) from inducing apoptosis in rat cardiomyocytes. Mechanistically, CT, PG, and RA induce increases of Bcl-xL protein and mRNA, and activate a 3.2 kb bcl-x gene promoter. CT and RA, but not PG, induced the activity of a 0.9 kb bcl-x promoter, containing sequences for AP-1 and NF-kB binding. RA, but not CT or PG, induced NF-kB activation. CT, but not PG or RA, induced AP-1 activation, and induction of the 0.9 kb bcl-x reporter by CT was inhibited by dominant negative c-Jun TAM-67 or removal of AP-1 binding site. Therefore, although CT, PG, and RA all induce Bcl-xL mRNA and protein, three independent mechanisms are in operation: while CT induces Bcl-xL via AP-1 transcription factor, and RA induces NF-kB activation and bcl-x promoter activity, PG induces Bcl-xL via a mechanism independent of NF-kB or AP-1.

Dodecafluoropentane emulsion elicits cardiac protection against myocardial infarction through an ATP-Sensitive K+ channel dependent mechanism.

PURPOSE: Dodecafluoropentane emulsion (DDFPe) is a perfluorocarbon with high oxygen dissolving, transport, and delivery capacity that may offer the potential to limit ischemic injury prior to clinical reperfusion. Here we investigated the cardiac protective potential of DDFPe in a mouse model of myocardial infarction. METHODS: Myocardial infarction was initiated by permanent ligation of the left anterior descending (LAD) coronary artery. Mice were administered vehicle or 5-hydroxydecanoate (5-HD) intravenously 10 min before LAD occlusion followed by a single intravenous administration of vehicle or DDFPe immediately after occlusion. Heart tissue and serum samples were collected 24 after LAD occlusion for measurement of infarct size and cardiac troponin I (cTnI) levels, respectively. RESULTS: DDFPe treatment reduced infarct size by approximately 72% (36.9 ± 4.2% for vehicle vs 10.4 ± 2.3% for DDFPe; p < 0.01; n = 6-8) at 24 h. Serum cTnI levels were similarly reduced by DDFPe (35.0 ± 4.6 ng/ml for vehicle vs 15.8 ± 1.6 ng/ml for DDFPe; p < 0.01; n = 6-8). Pretreatment with 5-HD, a mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) inhibitor, blocked the reduction in infarct size (29.2 ± 4.4% for 5-HD vs 35.4 ± 7.4% for 5-HD+DDFPe; p = 0.48; n = 6-8) and serum cTnI levels (27.4 ± 5.1 ng/ml for 5-HD vs 34.6 ± 5.3 ng/ml for 5-HD+DDFPe; p = 0.86; n = 6-8) by DDFPe. CONCLUSION: Our data indicate a cardiac protective role of DDFPe that persists beyond its retention time in the body and is dependent on mitoK(ATP), an important mediator of ischemic preconditioning induced cardiac protection.

Glucocorticoid induced leucine zipper inhibits apoptosis of cardiomyocytes by doxorubicin.

Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found glucocorticoid-induced leucine zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes.

NAD(P)H:quinone oxidoreductase 1 is induced by progesterone in cardiomyocytes.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of various quinones by utilizing NAD(P)H as an electron donor. Our previous study found that progesterone (PG) can protect cardiomyocytes from apoptosis induced by doxorubicin (Dox). Microarray analyses of genes induced by PG had led to the discovery of induction of NQO1 mRNA. We report here that PG induces NQO1 protein and its activity in a dose-dependent manner. Whereas NQO1 is well known as a target gene of Nrf2 transcription factor due to the presence of antioxidant response element (ARE) in the promoter, PG did not activate the ARE, suggesting Nrf2-independent induction of NQO1. To address the role of NQO1 induction in PG-induced cytoprotection, we tested the effect of NQO1 inducer ß-naphthoflavone and inhibitor dicoumarol. Induction of NQO1 by ß-naphthoflavone decreased Dox-induced apoptosis and potentiated the protective effect of PG as measured by caspase-3 activity. PG-induced NQO1 activity was inhibited with dicoumarol, which did not affect PG-induced cytoprotection. Dicoumarol treatment alone potentiated Dox-induced caspase-3 activity. These data suggest that while NQO1 plays a role in PG-induced cytoprotection, there are additional components contributing to PG-induced cytoprotection.

p21(WAF1/Cip1/Sdi1) knockout mice respond to doxorubicin with reduced cardiotoxicity.

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21(WAF1/Cip1/Sdi1) (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFNγ and TNFα in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice.

Dexamethasone induces transcriptional activation of Bcl-xL gene and inhibits cardiac injury by myocardial ischemia.

Psychological or physical stress causes an elevation of glucocorticoids in the circulating system. Glucocorticoids regulate a variety of physiological functions, from energy metabolism and biochemical homeostasis to immune response. Synthetic steroids are among the most prescribed drugs for immune suppression and chemotherapy. While glucocorticoids are best known for inducing apoptosis in a number of cell types, we have found that corticosteroids at stress relevant levels protect cardiomyocytes from apoptosis. Current study addresses whether glucocorticoids inhibit cardiac injury in vivo. Adult male C57BL6 mice were administered with dexamethasone (20mg/kg, i.p.) or vehicle control 20 h prior to left anterior descending coronary artery occlusion surgery. Myocardial infarction was measured by triphenyl tetrazoliumchloride staining in tissue slices and by levels of cardiac Troponin (cTn I) in the blood. Treatment of dexamethasone markedly reduced infarct size (19.6 ± 4.3%, vs. 29.2 ± 4.9%, p<0.01) and cTn I level in the blood (3.83 ± 0.66 ng/ml vs. 5.62 ± 0.37 ng/ml, p<0.01). In studying the mechanism of such protection, we found that dexamethasone induces the expression of Bcl-xL gene in the myocardium. With cardiomyocytes in culture, glucocorticoids increased transcription of Bcl-xL gene as evidenced by Bcl-xL mRNA increase and promoter activation. The glucocorticoid receptor antagonist mifepristone prevented dexamethasone from inducing cardiac protection or Bcl-xL expression. Our data suggest that activation of glucocorticoid receptor can prevent cardiac injury through transcriptional activation of Bcl-xL gene.

Inhibition of apoptosis by progesterone in cardiomyocytes.

While gender-based differences in heart disease have raised the possibility that estrogen (ES) or progesterone (PG) may have cardioprotective effects, recent controversy regarding hormone replacement therapy has questioned the cardiac effects of these steroids. Using cardiomyocytes, we tested whether ES or PG has protective effects at the cellular level. We found that PG but not ES protects cardiomyocytes from apoptotic cell death induced by doxorubicin (Dox). PG inhibited apoptosis in a dose-dependent manner, by 12 ± 4.0% at 1 µm and 60 ± 1.0% at 10 µm. The anti-apoptotic effect of PG was also time dependent, causing 18 ± 5% or 62 + 2% decrease in caspase-3 activity within 1 h or 72 h of pretreatment. While PG causes nuclear translocation of its receptor within 20 min, the cytoprotective effect of PG was canceled by mifepristone (MF), a PG receptor antagonist. Analyses using Affymetrix high-density oligonucleotide array and RT-PCR found that PG induced Bcl-xL, metallothionine, NADPH quinone oxidoreductase 1, glutathione peroxidase-3, and four isoforms of glutathione S-transferase. Western blot analyses revealed that PG indeed induced an elevation of Bcl-xL protein in a dose- and time-dependent manner. Nuclear run-on assay indicated that PG induced Bcl-xL gene transcription. Inhibiting the expression of Bcl-xL using siRNA reduced the cytoprotective effect of PG. Our data suggests that PG induces a cytoprotective effect in cardiomyocytes in association with induction of Bcl-xL gene.

Cystatin C increases in cardiac injury: a role in extracellular matrix protein modulation.

AIMS: Numerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology. METHODS AND RESULTS: Cardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H(2)O(2) for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H(2)O(2) caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III. CONCLUSION: Oxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.

Partial proteasome inhibition in human fibroblasts triggers accelerated M1 senescence or M2 crisis depending on p53 and Rb status.

Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.