David J. Granville

Allograft Vasculopathy Versus Atherosclerosis

Over the last 4 decades, heart transplantation (HTx) has evolved as a mainstream therapy for heart failure. Approximately half of patients needing HTx have organ failure consequent to atherosclerosis. Despite advances in immunosuppressive drugs, long-term success of HTx is limited by the development of a particular type of coronary atherosclerosis, referred to as cardiac allograft vasculopathy (CAV). Although the exact pathogenesis of CAV remains to be established, there is strong evidence that CAV involves immunologic mechanisms operating in a milieu of nonimmunologic risk factors. The immunologic events constitute the principal initiating stimuli, resulting in endothelial injury and dysfunction, altered endothelial permeability, with consequent myointimal hyperplasia and extracellular matrix synthesis. Lipid accumulation in allograft arteries is prominent, with lipoprotein entrapment in the subendothelial tissue, through interactions with proteoglycans. The apparent endothelial “intactness” in human coronary arteries of the transplanted heart suggest that permeability and function of the endothelial barrier altered. Various insults to the vascular bed result in vascular smooth muscle cell (SMC) activation. Activated SMCs migrate from the media into the intima, proliferate, and elaborate cytokines and extracellular matrix proteins, resulting in luminal narrowing and impaired vascular function. Arteriosclerosis is a broad term that is used to encompass all diseases that lead to arterial hardening, including native atherosclerosis, postangioplasty restenosis, vein bypass graft occlusion, and CAV. These diseases exhibit many similarities; however, they are distinct from one another in numerous ways as well. The present review summarizes the current understanding of the risk factors and the pathophysiological similarities and differences between CAV and atherosclerosis.

Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy

Photodynamic therapy (PDT) is a clinical approach that utilizes light‐activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT‐induced apoptosis are poorly defined. It has been shown for other pro‐apoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase‐3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT‐induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT‐treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1–2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2–3 h post PDT. The caspase‐3 inhibitor DEVD‐fmk blocked caspase‐8 and Bap31 cleavage suggesting that caspase‐8 and Bap31 processing occur downstream of caspase‐3 activation in PDT‐induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase‐8 activation and demonstrates that caspase‐8 activation can occur after cytochrome c release.

Inflammation in myocardial diseases.

Inflammatory processes underlie a broad spectrum of conditions that injure the heart muscle and cause both structural and functional deficits. In this article, we address current knowledge regarding 4 common forms of myocardial inflammation: myocardial ischemia and reperfusion, sepsis, viral myocarditis, and immune rejection. Each of these pathological states has its own unique features in pathogenesis and disease evolution, but all reflect inflammatory mechanisms that are partially shared. From the point of injury to the mobilization of innate and adaptive immune responses and inflammatory amplification, the cellular and soluble mediators and mechanisms examined in this review will be discussed with a view that both beneficial and adverse consequences arise in these human conditions.

Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma.

The cytotoxic granzyme B (GrB)/perforin pathway has been traditionally viewed as a primary mechanism that is used by cytotoxic lymphocytes to eliminate allogeneic, virally infected and/or transformed cells. Although originally proposed to have intracellular and extracellular functions, upon the discovery that perforin, in combination with GrB, could induce apoptosis, other potential functions for this protease were, for the most part, disregarded. As there are 5 granzymes in humans and 11 granzymes in mice, many studies used perforin knockout mice as an initial screen to evaluate the role of granzymes in disease. However, in recent years, emerging clinical and biochemical evidence has shown that the latter approach may have overlooked a critical perforin-independent, pathogenic role for these proteases in disease. This review focuses on GrB, the most characterized of the granzyme family, in disease. Long known to be a pro-apoptotic protease expressed by cytotoxic lymphocytes and natural killer cells, it is now accepted that GrB can be expressed in other cell types of immune and nonimmune origin. To the latter, an emerging immune-independent role for GrB has been forwarded due to recent discoveries that GrB may be expressed in nonimmune cells such as smooth muscle cells, keratinocytes, and chondrocytes in certain disease states. Given that GrB retains its activity in the blood, can cleave extracellular matrix, and its levels are often elevated in chronic inflammatory diseases, this protease may be an important contributor to certain pathologies. The implications of sustained elevations of intracellular and extracellular GrB in chronic vascular, dermatological, and neurological diseases, among others, are developing. This review examines, for the first time, the multiple roles of GrB in disease pathogenesis.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12−/− but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrates gene exons and by MMP-12–mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3–infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Mechanism of Colon Cancer Cell Apoptosis Mediated by Pyropheophorbide-a Methylester Photosensitization

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-κB activation and apoptosis were clearly independent although NF-κB was shown to counteract apoptosis mediated by PPME photosensitization.

Release of Cytochrome c, Bax Migration, Bid Cleavage, and Activation of Caspases 2, 3, 6, 7, 8, and 9 during Endothelial Cell Apoptosis

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.

Attenuated Acute Cardiac Rejection in NOS2 −/− Recipients Correlates With Reduced Apoptosis

BACKGROUND The mechanisms through which NOS2-mediated pathways regulate graft failure in acute cardiac rejection are ill defined. To determine whether apoptosis promoted by NOS2 may contribute, we used a heterotopic transplant model to study mouse cardiac allografts placed in recipients with targeted gene deletion of NOS2. METHODS AND RESULTS Using 5 different indexes of apoptosis, we showed that mouse cardiac allografts placed in NOS2 -/- recipients (n=7) had reduced apoptotic activity compared with those in NOS2 +/+ controls (n=8). There were significantly fewer TUNEL-positive nuclei per high-powered field (P<0.01), less DNA fragmentation (antinucleosome ELISA; P<0.05), lower corrected transcript levels for caspase-1 and -3 (32P reverse transcriptase-polymerase chain reaction; P<0.01), and reduced caspase-3 activity (cleavage of DEVD-pNA [P<0.001] and poly [ADP-ribose] polymerase) in grafts from NOS2 -/- recipients. This concordant reduction in apoptotic indexes paralleled the improved histological outcome of grafts transplanted into NOS2 -/- recipients (assessed as rejection scores; P=0.012). To identify pathways controlled by NOS2, we compared intragraft transcript levels of potential triggers and regulators. Whereas Fas ligand/Fas and tumor necrosis factor (TNF)-alpha/TNF receptor-1 levels were not altered by NOS2 deficiency, transcript levels for p53 were significantly lower in grafts from NOS2 -/- recipients, coinciding with a significant increase in the antiapoptotic Bcl-2/Bax balance and decrease in Bcl-Xl levels. CONCLUSIONS Using NOS2 knockout mice, we demonstrated that NOS2-mediated pathways can promote acute rejection, at least in part, by inducing apoptotic cell death. When NOS2 is present, p53 might control NOS2-mediated apoptosis by stimulating Bax and repressing Bcl-2 and Bcl-Xl expression, which may activate the cell death program in the rejecting heart.

Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin Involvement of Extracellular Matrix Degradation

Objective—T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death. Methods and Results—The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed, indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolytic activities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme B internalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMC extracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation. Conclusions—These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleaving extracellular proteins, such as fibronectin.

Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release.