Granzyme B Contributes to Choroidal Neovascularization and Age-Related Macular Degeneration Through Proteolysis of Thrombospondin-1.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. TSP-1 and TSP-2 proteolysis were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 and higher GzmB immunoreactivity. In CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

Perforin and granzyme B have separate and distinct roles during atherosclerotic plaque development in apolipoprotein E knockout mice.

The granzyme B/perforincytotoxic pathway is a well established mechanism of initiating target cell apoptosis. Previous studies have suggested a role for the granzyme B/perforin cytotoxic pathway in vulnerable atherosclerotic plaque formation. In the present study, granzyme B deficiency resulted in reduced atherosclerotic plaque development in the descending aortas of apolipoprotein E knockout mice fed a high fat diet for 30 weeks while perforindeficiency resulted in greater reduction in plaque development with significantly less plaque area than granzyme Bdeficient mice. In contrast to the descending aorta, no significant change in plaque size was observed in aortic roots from either granzyme Bdeficient or perforindeficient apolipoprotein E knockout mice. However, atherosclerotic plaques in the aortic roots did exhibit significantly more collagen in granzyme B, but not perforin deficient mice. Together these results suggest significant, yet separate roles for granzyme B and perforin in the pathogenesis of atherosclerosis that go beyond the traditional apoptotic pathway with additional implications in plaque development, stability and remodelling of extracellular matrix.

Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-ß1.

OBJECTIVE: Granzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-ß1 into the extracellular milieu. METHODS/RESULTS: Three extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-ß1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-ß1 release. Our data confirmed that GrB liberated TGF-ß1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-ß1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells. CONCLUSION: In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-ß1 from PGs.

Granzyme B contributes to extracellular matrix remodeling and skin aging in apolipoprotein E knockout mice.

Apolipoprotein E knockout (apoE-KO) mice have been utilized for decades as a model of atherosclerosis. However, in addition to atherosclerosis, apoE-KO mice develop extensive cutaneous xanthomatosis, accelerated skin aging and frailty when fed a high fat diet. Granzyme B (GrB) is a pro-apoptotic serine protease that has recently been shown to exhibit extracellular proteolytic activity in certain pathologies. In the present study, the role of GrB in skin aging and pathology was assessed using the apoE-KO model of skin aging. Male C57BL/6 wild type and apoE-KO mice were grown for 0, 5, 15 or 30 weeks on either a high fat (21.2% fat, 0.2% cholesterol) or regular chow diet (7% fat). ApoE/GrB double knockout (DKO) mice were also generated and assessed after being fed either diet for 30 weeks. Skin was removed from the mid to lower back and examined for age-related changes such as hair loss, skin thinning and collagen remodeling and disorganization. ApoE-KO mice exhibited signs of frailty, hair graying, hair loss, skin thinning, loss of collagen density and increased skin pathologies featuring collagen remodeling and reduced decorin compared to wild type controls. These phenotypes occurred earlier and were more severe when fed a high fat diet. In addition, we also observed increased GrB expression in proximity to areas of decorin degradation and reduced collagen density in the skin of apoE-KO mice. DKO mice exhibited protection against skin thinning, ECM degradation and loss of dermal collagen density. In summary, our results provide novel insights into the effects of a high fat diet and apoE deficiency on skin aging and pathology and suggest a role for GrB in age-related skin thinning and frailty.

Perforin-independent extracellular granzyme B activity contributes to abdominal aortic aneurysm.

Granzyme B (GZMB) is a serine protease that is abundantly expressed in advanced human atherosclerotic lesions and may contribute to plaque instability. Perforin is a pore-forming protein that facilitates GZMB internalization and the induction of apoptosis. Recently a perforin-independent, extracellular role for GZMB has been proposed. In the current study, the role of GZMB in abdominal aortic aneurysm (AAA) was assessed. Apolipoprotein E (APOE)(-/-) x GZMB(-/-) and APOE(-/-) x perforin(-/-) double knockout (GDKO, PDKO) mice were generated to test whether GZMB exerted a causative role in aneurysm formation. To induce aneurysm, mice were given angiotensin II (1000 ng/kg/min) for 28 days. GZMB was found to be abundant in both murine and human AAA specimens. GZMB deficiency was associated with a decrease in AAA and increased survival compared with APOE-KO and PDKO mice. Although AAA rupture was observed frequently in APOE-KO (46.7%; n = 15) and PDKO (43.3%; n = 16) mice, rupture was rarely observed in GDKO (7.1%; n = 14) mice. APOE-KO mice exhibited reduced fibrillin-1 staining compared with GDKO mice, whereas in vitro protease assays demonstrated that fibrillin-1 is a substrate of GZMB. As perforin deficiency did not affect the outcome, our results suggest that GZMB contributes to AAA pathogenesis via a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity.

Detection and quantification of apoptosis in the vasculature.

The integral role of apoptosis in the pathogenesis of cardiovascular diseases has been extensively studied and characterized in recent years. The study of cell death in the vasculature has significantly contributed to our knowledge of vascular disease pathology and has played a role in identifying potential therapeutic strategies for these diseases. This chapter describes a number of standard, widely used protocols for detecting and quantifying apoptosis in vessel wall cells and tissue. These techniques include terminal deoxynucleotidyl transferase dUTP nick-end labeling staining for DNA fragmentation, Hoechst staining for chromatin condensation, Annexin V staining, labeling for phosphatidylserine externalization, Western blot assessment of caspase cleavage, immunofluorescence detection of caspase activation, assessment of mitochondrial membrane depolarization and cytochrome c release, and a splenocyte assay for quantifying susceptibility to immune cell-mediated apoptosis.

Influence of interleukin-1alpha on androgen receptor expression and cytokine secretion by cultured human dermal papilla cells.

Dermal papilla cells (DPC) control the growth character of the hair follicle through their elaboration of mitogenic factors and extracellular matrix components. Further, the dermal papilla is a primary site of androgen action in the hair follicle. Interleukin-1alpha (IL-1alpha) is prominent in skin wounding and inflammatory responses although regarded as a negative hair growth regulator. We studied the effect of IL-1alpha and the potent androgen 5alpha-dihydrotestosterone (DHT) on the expression of the androgen receptor (AR) and various factors secreted by cultured human temporal scalp DPC. IL-1alpha triggered cellular changes consistent with nuclear factor-kappaB pathway activation as well as reduced AR mRNA and protein expression levels for DHT-stimulated DPC. This cytokine also increased DPC supernatant keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations. IL-1alpha did not influence DPC supernatant levels of transforming growth factor-beta1, a negative hair growth regulator. The stimulatory effect of IL-1alpha on DPC VEGF, GM-CSF, KGF, and IL-8 expression was also evident at the mRNA level for these cytokines. IL-1alpha also increased mRNA transcript levels of protease-nexin-1, a secreted serine protease inhibitor expressed in the dermal papilla of anagen-stage hair follicles. Although DHT did not affect supernatant cytokine concentrations, the androgen altered mRNA transcript levels of several factors for DPC co-stimulated with IL-1alpha. In consideration of its in vitro activity profile, IL-1alpha may be an important modifier of dermal papilla activity as well as potentially influence androgen-regulated gene expression in DPC.

Ultraviolet B light stimulates interleukin-20 expression by human epithelial keratinocytes.

The proinflammatory cytokine interleukin-20 (IL-20) may exert the majority of its activity in the skin. We examined the effect of various treatments including several forms of phototherapy on IL-20 expression using cultured normal human epithelial keratinocytes (NHEK). Broadband UVB light, recombinant (r) IL-1 and rIL-8 increased, while hydrocortisone reduced, NHEK supernatant IL-20 levels. Elevation of NHEK IL-20 mRNA and maximal supernatant IL-20 levels occurred with a UVB light dose (40 mJ cm(-2)) that reduced cell viability by approximately 50%. While this UVB light dose also elevated supernatant IL-1 alpha and IL-8 levels, antibody neutralization studies indicated that neither of these cytokines was directly responsible for this increase in IL-20 expression. However, the elevation in IL-20 levels was fully inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580, suggesting involvement of this stress signaling pathway in this UVB light response. Photodynamic therapy (PDT) with the photosensitizer lemuteporfin, UVA light, cisplatin, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) or recombinant interferon-gamma (rIFN-gamma) either had little effect or decreased NHEK supernatant IL-20 levels. Reduced IL-20 levels paralleled the cytotoxic actions of PDT, UVA light or cisplatin and the antiproliferative effect of rIFN-gamma. Neither rIL-20 supplementation nor anti-IL-20 antibody treatments affected cell viability indicating that soluble IL-20 did not affect the short-term survival of UVB light-irradiated NHEK. Stimulation of IL-20 expression in keratinocytes by UVB light suggests that this cytokine might participate in skin responses to this ever-present environmental factor and potentially has a role in UV light-associated dermatoses.