Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.

Cbx3 inhibits vascular smooth muscle cell proliferation, migration, and neointima formation.

AIMS: To investigate the role of chromobox protein homolog 3 (Cbx3) in vascular smooth muscle cell (VSMC) proliferation, migration, and neointima formation following vascular injury. METHODS AND RESULTS: Overexpression of Cbx3 led to a significant increase in VSMC contractile gene expression and VSMC apoptosis as well as a dramatic decrease in collagen gene expression, VSMC proliferation, and migration. Meanwhile, the opposite was observed following inhibition of endogenous Cbx3. Luciferase activity assays revealed that Notch signalling, but neither ß-catenin nor NF-κB signalling, is regulated by Cbx3 in VSMCs, and among the four Notch receptors, Notch3 is selectively down-regulated by Cbx3 through a transcriptional repression mechanism. Notch3 gene activation recapitulates the effects of Cbx3 knockdown on VSMC proliferation and migration. Consequently, the inhibitory effects of Cbx3 over-expression on VSMC proliferation and migration were reversed by Notch3 gene reactivation. In a model of vascular damage by carotid wire injury, we observed that Cbx3 expression was dramatically down-regulated in the injured arteries. Local ectopic over-expression of Cbx3 in the injured arteries significantly inhibited Notch3 expression, thereby reducing VSMCs proliferation and causing an overall decrease in neointima formation. Additionally, injury-induced neointimal SMC hyperplasia was significantly reduced by aortic inhibition of Notch3. Importantly, a decreased expression level of Cbx3, but an increased expression level of Notch3, was observed in human femoral arteries with atherosclerotic lesions. CONCLUSION: Cbx3 modulates VSMC contractile and collagen gene expression, as well as VSMC proliferation, migration, and apoptosis via a Notch3 pathway, and plays an important role in controlling injury-induced neointima formation.

miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation.

BACKGROUND: MicroRNA-22 (miR-22) has recently been reported to play a regulatory role during vascular smooth muscle cell (VSMC) differentiation from stem cells, but little is known about its target genes and related pathways in mature VSMC phenotypic modulation or its clinical implication in neointima formation following vascular injury. METHODS: We applied a wire-injury mouse model, and local delivery of AgomiR-22 or miR-22 inhibitor, as well, to explore the therapeutic potential of miR-22 in vascular diseases. Furthermore, normal and diseased human femoral arteries were harvested, and various in vivo, ex vivo, and in vitro models of VSMC phenotype switching were conducted to examine miR-22 expression during VSMC phenotype switching. RESULTS: Expression of miR-22 was closely regulated during VSMC phenotypic modulation. miR-22 overexpression significantly increased expression of VSMC marker genes and inhibited VSMC proliferation and migration, whereas the opposite effect was observed when endogenous miR-22 was knocked down. As expected, 2 previously reported miR-22 target genes, MECP2 (methyl-CpG binding protein 2) and histone deacetylase 4, exhibited a regulatory role in VSMC phenotypic modulation. A transcriptional regulator and oncoprotein, EVI1 (ecotropic virus integration site 1 protein homolog), has been identified as a novel miR-22 target gene in VSMC phenotypic modulation. It is noteworthy that overexpression of miR-22 in the injured vessels significantly reduced the expression of its target genes, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries, whereas the opposite effect was observed with local application of a miR-22 inhibitor to injured arteries. We next examined the clinical relevance of miR-22 expression and its target genes in human femoral arteries. We found that miR-22 expression was significantly reduced, whereas MECP2 and EVI1 expression levels were dramatically increased, in diseased in comparison with healthy femoral human arteries. This inverse relationship between miR-22 and MECP2 and EVI1 was evident in both healthy and diseased human femoral arteries. CONCLUSIONS: Our data demonstrate that miR-22 and EVI1 are novel regulators of VSMC function, specifically during neointima hyperplasia, offering a novel therapeutic opportunity for treating vascular diseases.

Novel Pathological Role of hnRNPA1 (Heterogeneous Nuclear Ribonucleoprotein A1) in Vascular Smooth Muscle Cell Function and Neointima Hyperplasia.

OBJECTIVE: hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) plays a variety of roles in gene expression. However, little is known about the functional involvement of hnRNPA1 in vascular smooth muscle cell (VSMC) function and neointima hyperplasia. In this study, we have attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC function, injury-induced vessel remodeling, and human atherosclerotic lesions, as well as discern the molecular mechanisms involved. APPROACH AND RESULTS: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation, and migration were regulated by hnRNPA1. Our data show that hnRNPA1 exerts its effects on VSMC functions through modulation of IQGAP1 (IQ motif containing GTPase activating protein 1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through 2 mechanisms: upregulating microRNA-124 (miR-124) and binding to AU-rich element of IQGAP1 gene. Further evidence suggests that hnRNPA1 upregulates miR-124 by modulating miR-124 biogenesis and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. CONCLUSIONS: Our data show that hnRNPA1 is a critical regulator of VSMC function and behavior in the context of neointima hyperplasia, and the hnRNPA1/miR-124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.

NCK Associated Protein 1 Modulated by miRNA-214 Determines Vascular Smooth Muscle Cell Migration, Proliferation, and Neointima Hyperplasia.

BACKGROUND: MicroRNA miR-214 has been implicated in many biological cellular functions, but the impact of miR-214 and its target genes on vascular smooth muscle cell (VSMC) proliferation, migration, and neointima smooth muscle cell hyperplasia is unknown. METHODS AND RESULTS: Expression of miR-214 was closely regulated by different pathogenic stimuli in VSMCs through a transcriptional mechanism and decreased in response to vascular injury. Overexpression of miR-214 in serum-starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miR-214 dramatically increased VSMC proliferation and migration. Gene and protein biochemical assays, including proteomic analyses, showed that NCK associated protein 1 (NCKAP1)-a major component of the WAVE complex that regulates lamellipodia formation and cell motility-was negatively regulated by miR-214 in VSMCs. Luciferase assays showed that miR-214 substantially repressed wild-type but not the miR-214 binding site mutated version of NCKAP1 3' untranslated region luciferase activity in VSMCs. This result confirmed that NCKAP1 is the functional target of miR-214 in VSMCs. NCKAP1 knockdown in VSMCs recapitulates the inhibitory effects of miR-214 overexpression on actin polymerization, cell migration, and proliferation. Data from cotransfection experiments also revealed that inhibition of NCKAP1 is required for miR-214-mediated lamellipodia formation, cell motility, and growth. Importantly, locally enforced expression of miR-214 in the injured vessels significantly reduced NCKAP1 expression levels, inhibited VSMC proliferation, and prevented neointima smooth muscle cell hyperplasia after injury. CONCLUSIONS: We uncovered an important role of miR-214 and its target gene NCKAP1 in modulating VSMC functions and neointima hyperplasia. Our findings suggest that miR-214 represents a potential therapeutic target for vascular diseases.

miRNA-34a reduces neointima formation through inhibiting smooth muscle cell proliferation and migration.

AIMS: We have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells in vitro and in vivo. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. METHODS AND RESULTS: Various pathological stimuli regulate miR-34a expression in VSMCs through a transcriptional mechanism, and the P53 binding site is required for miR-34a gene regulation by these stimuli. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodeling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1-3'-UTR-luciferase activity in VSMCs, but not mutant Notch1-3'-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, the expression level of miR-34a was significantly down-regulated in injured arteries, and miR-34a perivascular over-expression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. CONCLUSION: Our data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.

A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization.

Matrix metalloproteinase-8 (MMP8) has been shown to influence various cellular functions. As monocytes and macrophages (Mφ) express MMP8, we investigated if MMP8 played a role in macrophage differentiation and polarization. MMP8 expression was significantly increased during monocyte differentiation into Mφ. Monocyte-derived Mφ from MMP8-deficient mice expressed higher levels of M1-Mφ markers but lower levels of M2-Mφ markers than monocyte-derived Mφ from wild-type mice. Although Mφ from either MMP8-deficient or wild-type mice were inducible by interferon-γ into M1-Mφ, only wild-type Mφ but not MMP8-deficient Mφ could be induced into M2-Mφ by interleukin-4. However, MMP8-deficient Mφ exposed to conditioned culture media of wild-type Mφ developed a M2-Mφ phenotype. Compared with conditioned culture media of wild-type Mφ, conditioned culture media of MMP8-deficient Mφ contained a lower concentration of active transforming growth factor-ß (TGF-ß), an M2-Mφ inducer. Moreover, evidence also showed that the degradation of the TGF-ß sequester, fibromodulin, was modulated by MMP8. The data indicate a previously unknown role of MMP8 in M2-Mφ polarization by cleaving fibromodulin and therefore increasing the bioavailability of the M2-Mφ inducer TGF-ß.

MicroRNA-22 regulates smooth muscle cell differentiation from stem cells by targeting methyl CpG-binding protein 2.

OBJECTIVE: In this study, we attempted to uncover the functional impact of microRNA-22 (miR-22) and its target gene in smooth muscle cell (SMC) differentiation and delineate the molecular mechanism involved. APPROACH AND RESULTS: miR-22 was found to be significantly upregulated during SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells. Enforced expression of miR-22 by its mimic, while knockdown of miR-22 by its antagomiR, promotes or inhibits SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells, respectively. Expectedly, miR-22 overexpression in stem cells promoted SMC differentiation in vivo. Methyl CpG-binding protein 2 (MECP2) was predicted as one of the top targets of miR-22. Interestingly, the gene expression levels of MECP2 were significantly decreased during SMC differentiation, and MECP2 was dramatically decreased in miR-22 overexpressing cells but significantly increased when miR-22 was knockdown in the differentiating stem cells. Importantly, luciferase assay showed that miR-22 substantially inhibited wild-type, but not mutant MECP2-3' untranslated region-luciferase activity. In addition, modulation of MECP2 expression levels affects multiple SMC-specific gene expression in differentiated embryonic stem cells. Mechanistically, our data showed that MECP2 could transcriptionally repress SMC gene expression through modulating various SMC transcription factors, as well as several proven SMC differentiation regulators. Evidence also revealed that enrichment of H3K9 trimethylation around the promoter regions of the SMC differentiation regulators genes were significantly increased by MECP2 overexpression. Finally, miR-22 was upregulated by platelet-derived growth factor-BB and transforming growth factor-ß through a transcriptional mechanism during SMC differentiation. CONCLUSIONS: miR-22 plays an important role in SMC differentiation, and epigenetic regulation through MECP2 is required for miR-22 mediated SMC differentiation.

Disturbed blood flow induces RelA expression via c-Jun N-terminal kinase 1: a novel mode of NF-κB regulation that promotes arterial inflammation.

RATIONALE: The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. OBJECTIVE: We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. METHODS AND RESULTS: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. CONCLUSIONS: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK-NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

Dexamethasone arterializes venous endothelial cells by inducing mitogen-activated protein kinase phosphatase-1: a novel antiinflammatory treatment for vein grafts?

BACKGROUND: Vein grafting in coronary artery surgery is complicated by a high restenosis rate resulting from the development of vascular inflammation, intimal hyperplasia, and accelerated atherosclerosis. In contrast, arterial grafts are relatively resistant to these processes. Vascular inflammation is regulated by signaling intermediaries, including p38 mitogen-activated protein (MAP) kinase, that trigger endothelial cell (EC) expression of chemokines (eg, interleukin-8, monocyte chemotactic protein-1) and other proinflammatory molecules. Here, we have tested the hypothesis that p38 MAP kinase activation in response to arterial shear stress (flow) may occur more readily in venous ECs, leading to greater proinflammatory activation. METHODS AND RESULTS: Comparative reverse-transcriptase polymerase chain reaction and Western blotting revealed that arterial shear stress induced p38-dependent expression of monocyte chemotactic protein-1 and interleukin-8 in porcine jugular vein ECs. In contrast, porcine aortic ECs were protected from shear stress-induced expression of p38-dependent chemokines as a result of rapid induction of MAP kinase phosphatase-1. However, we observed with both cultured porcine jugular vein ECs and perfused veins that venous ECs can be protected by brief treatment with dexamethasone, which induced MAP kinase phosphatase-1 to suppress proinflammatory activation. CONCLUSIONS: Arterial but not venous ECs are protected from proinflammatory activation in response to short-term exposure to high shear stress by the induction of MAP kinase phosphatase-1. Dexamethasone pretreatment arterializes venous ECs by inducing MAP kinase phosphatase-1 and may protect veins from inflammation.

c-Jun N-terminal kinase primes endothelial cells at atheroprone sites for apoptosis.

OBJECTIVE: Atherosclerosis is a focal disease that occurs predominantly at branches and bends of the arterial tree. Endothelial cells (EC) at atherosusceptible sites are prone to injury, which can contribute to lesion formation, whereas EC at atheroprotected sites are resistant. The c-Jun N-terminal kinase (JNK) is activated constitutively in EC at atherosusceptible sites but is inactivated at atheroprotected sites by mitogen-activated protein kinase phosphatase-1 (MKP-1). Here, we examined the effects of JNK activation on EC physiology at atherosusceptible sites. METHODS AND RESULTS: We identified transcriptional programs regulated by JNK by applying a specific pharmacological inhibitor to cultured EC and assessing the transcriptome using microarrays. This approach and subsequent validation by gene silencing revealed that JNK positively regulates the expression of numerous proapoptotic molecules. Analysis of aortae of wild-type, JNK1(-/-), and MKP-1(-/-) mice revealed that EC at an atherosusceptible site express proapoptotic proteins and are primed for apoptosis and proliferation in response to lipopolysaccharide through a JNK1-dependent mechanism, whereas EC at a protected site expressed lower levels of proapoptotic molecules and were protected from injury by MKP-1. CONCLUSIONS: Spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries.

Activation of Nrf2 in endothelial cells protects arteries from exhibiting a proinflammatory state.

OBJECTIVE: Proinflammatory mediators influence atherosclerosis by inducing adhesion molecules (eg, VCAM-1) on endothelial cells (ECs) via signaling intermediaries including p38 MAP kinase. Regions of arteries exposed to high shear stress are protected from inflammation and atherosclerosis, whereas low-shear regions are susceptible. Here we investigated whether the transcription factor Nrf2 regulates EC activation in arteries. METHODS AND RESULTS: En face staining revealed that Nrf2 was activated in ECs at an atheroprotected region of the murine aorta where it negatively regulated p38-VCAM-1 signaling, but was expressed in an inactive form in ECs at an atherosusceptible site. Treatment with sulforaphane, a dietary antioxidant, activated Nrf2 and suppressed p38-VCAM-1 signaling at the susceptible site in wild-type but not Nrf2(-/-) animals, indicating that it suppresses EC activation via Nrf2. Studies of cultured ECs revealed that Nrf2 inactivates p38 by suppressing an upstream activator MKK3/6 and by enhancing the activity of the negative regulator MKP-1. CONCLUSIONS: Nrf2 prevents ECs at the atheroprotected site from exhibiting a proinflammatory state via the suppression of p38-VCAM-1 signaling. Pharmacological activation of Nrf2 reduces EC activation at atherosusceptible sites and may provide a novel therapeutic strategy to prevent or reduce atherosclerosis.

Increased endothelial mitogen-activated protein kinase phosphatase-1 expression suppresses proinflammatory activation at sites that are resistant to atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of arteries. It is triggered by proinflammatory mediators which induce adhesion molecules (eg, vascular cell adhesion molecule [VCAM]-1) in endothelial cells (ECs) by activating p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases by phosphorylation. Blood flow influences atherosclerosis by exerting shear stress (mechanical drag) on the inner surface of arteries, a force that alters endothelial physiology. Regions of the arterial tree exposed to high shear are protected from endothelial activation, inflammation, and atherosclerosis, whereas regions exposed to low or oscillatory shear are susceptible. We examined whether MAP kinase phosphatase (MKP)-1, a negative regulator of p38 and JNK, mediates the antiinflammatory effects of shear stress. We observed that expression of MKP-1 in cultured ECs was elevated by shear stress, whereas the expression of VCAM-1 was reduced. MKP-1 induction was shown to be necessary for the antiinflammatory effects of shear stress because gene silencing of MKP-1 restored VCAM-1 expression in sheared ECs. Immunostaining revealed that MKP-1 is preferentially expressed by ECs in a high-shear, protected region of the mouse aorta and is necessary for suppression of EC activation at this site, because p38 activation and VCAM-1 expression was enhanced by genetic deletion of MKP-1. We conclude that MKP-1 induction is required for the antiinflammatory effects of shear stress. Thus, our findings reveal a novel molecular mechanism contributing to the spatial distribution of vascular inflammation and atherosclerosis.

Elevated p53 expression is associated with dysregulation of the ubiquitin-proteasome system in dilated cardiomyopathy.

AIMS: The molecular mechanisms that regulate cardiomyocyte apoptosis and their role in human heart failure (HF) are uncertain. Expression of the apoptosis regulator p53 is governed by minute double minute 2 (MDM2), an E3 enzyme that targets p53 for ubiquitination and proteasomal processing, and by the deubiquitinating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP), which rescues p53 by removing ubiquitin chains from it. Here, we examined whether elevated expression of p53 was associated with dysregulation of ubiquitin-proteasome system (UPS) components and activation of downstream effectors of apoptosis in human dilated cardiomyopathy (DCM). METHODS AND RESULTS: Left ventricular myocardial samples were obtained from patients with DCM (n = 12) or from non-failing (donor) hearts (n = 17). Western blotting and immunohistochemistry revealed that DCM tissues contained elevated levels of p53 and its regulators MDM2 and HAUSP (all P < 0.01) compared with non-failing hearts. DCM tissues also contained elevated levels of polyubiquitinated proteins and possessed enhanced 20S-proteasome chymotrypsin-like activities (P < 0.04) as measured in vitro using a fluorogenic substrate. DCM tissues contained activated caspases-9 and -3 (P < 0.001) and reduced expression of the caspase substrate PARP-1 (P < 0.05). Western blotting and immunohistochemistry revealed that DCM tissues contained elevated expression levels of caspase-3-activated DNAse (CAD; P < 0.001), which is a key effector of DNA fragmentation in apoptosis and also contained elevated expression of a potent inhibitor of CAD (ICAD-S; P < 0.01). CONCLUSION: Expression of p53 in human DCM is associated with dysregulation of UPS components, which are known to regulate p53 stability. Elevated p53 expression and caspase activation in DCM was not associated with activation of both CAD and its inhibitor, ICAD-S. Our findings are consistent with the concept that apoptosis may be interrupted and therefore potentially reversible in human HF.