Lack of Proteinase 3 Stabilizes Advanced Atherosclerotic Lesions

Cardiovascular diseases are the leading cause of mortality in industrialized countries, representing 31% of deaths worldwide. The rupture of advanced atherosclerotic lesions leads to clinical manifestations such as myocardial infarction or stroke and possibly lethal clinical outcomes. The relevance of neutrophils in atherosclerosis has been neglected for a long time; however, extensive research during the last decade has acknowledged their contribution to this disease.[1] Neutrophil serine proteases (NSPs), namely cathepsin G (CTSG), neutrophil elastase (NE), and proteinase 3 (PRTN3), are critical for the effective functioning of neutrophils, not only to combat infections but also during sterile inflammation. The implication of serine proteases in atherosclerosis has been tackled by several studies, which have frequently generated conflicting outcomes.[2] [3] [4] Cointerpretation of these studies is often problematic due to inconsistencies in the atherosclerosis models employed, which calls for a standardized examination of the role of NSPs in atherosclerosis. In the present study, we aimed at shedding light on the topic by using the same model of advanced atherosclerosis in Ctsg^−/− , Elane^−/− , and Elane^−/−Prtn3^−/− mice crossed onto an Apoe^−/− background and comparing traits of instability of the resulting lesions side-by-side. For this purpose, Apoe^−/− , Apoe^−/−Ctsg^−/−Apoe^−/−Elane^−/− , and Apoe^−/−Elane^−/−Prtn3^−/− mice were fed a high-fat diet (HFD) for 16 weeks and the plaque composition and stability were assessed upon histological (hematoxylin and eosin, Oil-RedO, Picosirius Red, and terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) and immunohistochemical staining (macrophages: Mac-2 and smooth muscle actin: αSMA) of aortic root cross-sections. A period of 16 weeks of diet was chosen due to the features of the plaque at this stage, where lesions appear complex with inflammatory macrophages, a necrotic core, and smooth muscle cells (SMCs) outlining the fibrous cap.[5] [6] These elements are key parameters to evaluate the morphology and instability of advanced lesions. The vulnerability index (VI) was calculated as previously reported.[7] Briefly, the ratio between unstable (U) and stable (S) features was determined, where U includes the sum of necrotic core area (% of the lesion area), Mac2⁺ cells (% of total DAPI⁺ cells), and of TUNEL⁺ cells (% of total DAPI⁺ cells) and S consists of SMA⁺ area (% of the lesion area) and collagen⁺ area (% of the lesion area).

Apoe^−/− , Apoe^−/−Elane^−/− , and Apoe^−/−Elane^−/−Prtn3^−/− mice presented similar plaque areas and lesional lipid content ([Fig. 1A–C]). Consistent with our previous work in early atherosclerosis,[8] Apoe^−/−Ctsg^−/− mice developed smaller lesions than the control group. Plaque rupture typically occurs in the thinnest point of the fibrous cap[9] and therefore the assessment of fibrous cap thickness is a surrogate parameter for plaque instability. NE-deficient mice exhibited thinner caps than the control group, which would suggest that these lesions are more prone to rupture. Interestingly, additional PRTN3 absence (Apoe^−/−Elane^−/−Prtn3^−/− ) resulted in increased fibrous cap thickness, indicating a compensatory effect to NE deficiency ([Fig. 1D]). These results indicate that despite the apparently homogeneous plaque size, the analysis of instability parameters might entail unalike contributions of NSPs to the pathophysiology of atherosclerosis.

Fig. 1 Characterization of advanced atherosclerosis in neutrophil serine protease (SNP)-deficient mice. Apoe ^−/−, Apoe ^−/− Ctsg ^−/−, Apoe ^−/− Elane ^−/−, and Apoe^−/−Elane^−/−Prtn3^−/− mice were fed a high-fat diet for 16 weeks and their aortic roots where analyzed.(A) Representative images of Oil-Red-O staining. (B) Quantification of Oil-Red-O-positive area, as percentage of lesion area. (C) Quantification of plaque size (as percentage of lesion area) by hematoxylin and eosin (H&E) staining. (D) Quantification of fibrous cap of atherosclerotic lesions after H&E staining. Representative images and quantification of necrotic core (as percentage of the lesion area) by H&E staining (E), apoptotic cells (as percentage of DAPI⁺ cells) by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (F), monocyte/macrophages (as percentage of DAPI⁺ cells) by Mac2 staining (G), smooth muscle actin (αSMA) area (as percentage of total lesion area) (H), and collagen content (as percentage of total lesion area) by PicosiriusRed staining (I). (J) Representation of the average plaque composition in aortic roots. Stabilizing parameters are shown in green tones and destabilizing parameters in red tones (K). Calculation of the vulnerability index by the formula VI = (%Mac2⁺ cells) + (% Apoptotic cells) + (% Necrotic core) / (% Collagen) + (% αSMA). Scale bar 200µm. ***p < 0.0005, **p < 0.005, and *p < 0.05 as calculated by one-way analysis of variance (ANOVA)Kruskal–Wallis with Dunn's posttest.

The vulnerability of the plaque is determined by the balance of destabilizing and stabilizing structural features. While large necrotic cores, accumulation of apoptotic cells, and inflammatory macrophages are considered destabilizing factors, higher SMC and collagen content are thought to stabilize atherosclerotic lesions. We have found that indicators of cell death in the lesion (necrotic core size and amount of TUNEL-positive cells) were remarkably lower in Apoe^−/−Elane^−/−Prtn3^−/− mice ([Fig. 1E], [F]). Noteworthy, macrophage ([Fig. 1G]) and neutrophil counts (data not shown) in the plaque showed no significant differences between the groups. Interestingly, stability-promoting parameters such as collagen and smooth muscle actin content were increased in PRNT3-deficient mice, while no changes were found in the other groups ([Fig. 1H], [I]).

To date, a knockout mouse model with single PRTN3 deficiency is not available. For this reason, it is important to assess the relevance of the presented results as an integrated comparative study. Nevertheless, overlapping contributions of NE over PRTN3 cannot be directly ruled out. Yet, the outstanding differences between the double NE/PRTN3 over the single NE knockout in terms of plaque stability strongly suggest that the involvement of PRTN3 is decidedly superior to that of NE. Taking these results together, the overall plaque composition (represented in [Fig. 1J] with red tones for destabilizing and green for stabilizing parameters) indicates that more than half of the Apoe^−/−Elane^−/−Prtn3^−/− plaque is constituted by stabilizing components, while Apoe^−/− , Apoe^−/−Ctsg^−/− , and Apoe^−/−Elane^−/− plaques show a more vulnerable composition. To assess the overall vulnerability relayed by these phenotypic differences, we calculated the vulnerability plaque index (VI).[7] Herein, we found that Apoe^−/−Elane^−/−Prtn3^−/− mice exhibit strikingly reduced plaque vulnerability indices, while no changes were found for Apoe^−/−Ctsg^−/− and Apoe^−/−Elane^−/− mice. These findings demonstrate that the presence of PRTN3, unlike CTSG and NE, is a destabilizing factor in advanced stages of atherosclerosis.

NE, CTSG, and PRTN3 belong to the group of NSPs enriched in neutrophil granules and contribute not only to modulate the immune response, but also to the pathophysiology of inflammatory diseases.[10] [11] In the last years, several studies have investigated the importance of one or another NSP in atherosclerosis. However, the conclusions of these works are often not comparable due to important differences in the atherosclerosis models used in every study. For instance, NE deficiency has recently been reported to lead to diminished plaque engrossment and increased signs of stability at 12 weeks of HFD.[2] Another study comparing the development of atherosclerosis in Apoe^−/− and Apoe^−/−Elane^−/−Prtn3^−/− mice concluded that mice deficient in both NE and PRTN3 exhibit smaller plaques at early (4 weeks of HFD) and advanced (8 weeks of HFD) stages.[4] While our previous observations coincide with smaller lesions after 4 weeks of HFD in Apoe^−/−Elane^−/−Prtn3^−/− mice,[3] this difference is no longer sustained at advanced phases (16 weeks of HFD),[3] which is corroborated by the results in the present study. This inconsistency might be accounted for the additional 8 weeks of HFD that our advanced atherosclerosis groups take. Thus, at 8 weeks of HFD, the reduced plaque size in Apoe^−/−Elane^−/−Prtn3^−/− mice reported at early stages[4] may still be appreciable.

Regarding CTSG, while we previously reported important differences at early stages of the disease in its absence,[8] other researchers only find them at advanced phases.[12] Discrepancies among these conclusions are most likely due to the fact that these studies often focus on different models of atherosclerosis (low-density lipoprotein knockout vs. apolipoprotein E knockout), time and composition of the HFD (e.g., 4, 12, 16, or 24 weeks), and assessment of atherosclerosis extension/stability in different aortic areas (aortic root vs. aortic arch). Given the disparity of reports, the present work provides a comparative survey where the role of the serine proteases in advanced atherosclerosis is studied under the same circumstances, thus providing a valuable contrasting tool to shed light on the matter.

The lesions in PRTN3/NE-deficient mice presented lower vulnerability in part due to an increase of stabilizing factors like the content of collagen and SMCs. In this context, it is known that PRTN3 can cleave structural components of the extracellular matrix including elastin, fibronectin, vitronectin, laminin, and collagen.[13] Along the same lines, PRTN3 has been described to process the human cathelicidin antimicrobial peptide hCAP-18, generating a fragment named LL-37,[14] which has been found to induce SMC death.[15] Of note, several proatherosclerotic functions have been attributed to the processed form of hCAP18 in mice,[16] [17] [18] which might contribute to explain the plaque stability conferred by lack of PRTN3 as observed in our study.

Among the crucial functions of the NSPs, processing of cytokines is of great importance. At a molecular level, there is a certain degree of redundancy between NE, CTSG, and PRTN3. However, specific biological substrates for these proteases have been described over the decades.[19] Particularly interesting is the case of interleukin (IL)-1β, a proinflammatory cytokine with remarkable implications in atherosclerosis.[20] In humans, PRTN3 but not NE or CTSG cleaves its pro-form and activates IL-1β. The explanation for this specificity probably resides on the distribution of negative and positive charges of the residues of IL-1β, making it suitable for the cleaving site of PRTN3 but not NE or CTSG.[21] Besides IL-1β, PRTN3 is involved in the activation and processing of several proinflammatory cytokines relevant in atherosclerosis, such as tumor necrosis factor-α, monocyte chemoattractant protein-1, and IL-8 and also cleaves and inactivates the atherosclerosis-restraining molecule Annexin A1.[22] [23] [24] [25] Importantly, PRTN3 cleaves and activates caspase-3, a mediator of apoptosis independent from caspase-8 and caspase-9, contributing to elevate the apoptotic cell mass.[26] In mice, the proinflammatory signature in macrophages following the phagocytosis of apoptotic cells expressing PRTN3 has been shown to be strictly dependent on the presence of this protein at the plasma membrane during apoptosis.[27] Thus, given the decreased size of the necrotic core that we find in atherosclerotic lesions from PRTN3/NE but no NE alone or CTSG-deficient mice, it is legitimate to hypothesize that PRTN3 exacerbates the proinflammatory environment and stimulates apoptotic cell accumulation.

In conclusion, we here present a descriptive analysis that highlights the importance of PRTN3 over the other NSPs to instill instability in advanced stages of atherosclerosis.

• References

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