Enhancing the Drug-Like Profile of a Potent Peptide PACE4 Inhibitor by the Formation of a Host-Guest Inclusion Complex with ß-Cyclodextrin.

The enzyme PACE4 has been validated as a promising therapeutic target to expand the range of prostate cancer (PCa) treatments. In recent years, we have developed a potent peptidomimetic inhibitor, namely, compound C23 (Ac-(DLeu)LLLRVK-4-amidinobenzylamide). Like many peptides, C23 suffers from an unfavorable drug-like profile which, despite our efforts, has not yet benefited from the usual SAR studies. Hence, we turned our attention toward a novel formulation strategy, i.e., the use of cyclodextrins (CDs). CDs can benefit compounds through the formation of "host-guest" complexes, shielding the guest from degradation and enhancing biological survival. In this study, a series of ßCD-C23 complexes have been generated and their properties evaluated, including potency toward the enzyme in vitro, a cell-based proliferation assay, and stability in plasma. As a result, a new ßCD-formulated lead compound has been identified, which, in addition to being more soluble and more potent, also showed an improved stability profile.

Cleavage of the V-ATPase associated prorenin receptor is mediated by PACE4 and is essential for growth of prostate cancer cells.

Phosphatase and tensin homolog (PTEN) mutation is common in prostate cancer during progression to metastatic and castration resistant forms. We previously reported that loss of PTEN function in prostate cancer leads to increased expression and secretion of the Prorenin Receptor (PRR) and its soluble processed form, the soluble Prorenin Receptor (sPRR). PRR is an essential factor required for proper assembly and activity of the vacuolar-ATPase (V-ATPase). The V-ATPase is a rotary proton pump required for the acidification of intracellular vesicles including endosomes and lysosomes. Acidic vesicles are involved in a wide range of cancer related pathways such as receptor mediated endocytosis, autophagy, and cell signalling. Full-length PRR is cleaved at a conserved consensus motif (R-X-X-R↓) by a member of the proprotein convertase family to generate sPRR, and a smaller C-terminal fragment, designated M8.9. It is unclear which convertase processes PRR in prostate cancer cells and how processing affects V-ATPase activity. In the current study we show that PRR is predominantly cleaved by PACE4, a proprotein convertase that has been previously implicated in prostate cancer. We further demonstrate that PTEN controls PRR processing in mouse tissue and controls PACE4 expression in prostate cancer cells. Furthermore, we demonstrate that PACE4 cleavage of PRR is needed for efficient V-ATPase activity and prostate cancer cell growth. Overall, our data highlight the importance of PACE4-mediated PRR processing in normal physiology and prostate cancer tumorigenesis.

Efficacy of PACE4 pharmacotherapy in JHU-LNCaP-SM preclinical model of androgen independent prostate cancer.

Prostate cancer (PCa) is a complex disease progressing from in situ to invasive or metastatic tumors while also being capable of modulating its androgen dependence. Understanding how novel therapies are working across the different stages of the disease is critical for their proper positioning in the spectrum of PCa treatments. The targeting of proprotein convertase PACE4 (Paired basic Amino Acid-Cleaving Enzyme 4) has been proposed as a novel approach to treat PCa. Animal studies performed on LNCaP xenografts, an androgen-dependent model, already yielded positive results. In this study, we tested PACE4 inhibition on JHU-LNCaP-SM, a newly described androgen-independent model, in cell-based and xenograft assays. Like LNCaP, JHU-LNCaP-SM cells express PACE4 and its oncogenic isoform PACE4-altCT. Using isoform-specific siRNAs, downregulation of PACE4-altCT resulted in JHU-LNCaP-SM growth inhibition. Furthermore, JHU-LNCaP-SM responded to the PACE4 pharmacological inhibitor known as C23 in cell-based assays as well as in athymic nude mice xenografts. These data support the efficacy of PACE4 inhibitors against androgen independent PCa thereby demonstrating that PACE4 is a key target in PCa. The JHU-LNCaP-SM cell line represents a model featuring important aspects of androgen-independent PCa, but it also represents a very convenient model as opposed to LNCaP cells for in vivo studies, as it allows rapid screening due to its high implantation rate and growth characteristics as xenografts.

Nanobodies identify an activated state of the TRIB2 pseudokinase.

Tribbles proteins (TRIB1-3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function.

A risk model for digital breast tomosynthesis to predict breast cancer and guide clinical care.

Screening with digital breast tomosynthesis (DBT) improves breast cancer detection and reduces false positives. However, currently, no breast cancer risk model takes advantage of the additional information generated by DBT imaging for breast cancer risk prediction. We developed and internally validated a DBT-based short-term risk model for predicting future late-stage and interval breast cancers after negative screening exams. We included the available 805 incident breast cancers and a random sample of 5173 healthy women matched on year of study entry in a nested case-control study from 154,200 multiethnic women, aged 35 to 74, attending DBT screening in the United States between 2014 and 2019. A relative risk model was trained using elastic net logistic regression and nested cross-validation to estimate risks for using imaging features and age. An absolute risk model was developed using derived risks and U.S. incidence and competing mortality rates. Absolute risks, discrimination performance, and risk stratification were estimated in the left-out validation set. The discrimination performance of 1-year risk was 0.82 (95% CI, 0.79 to 0.85) with good calibration (P = 0.7). Using the U.S. Preventive Service Task Force guidelines, 14% of the women were at high risk, 19.6 times higher compared to general risk. In this high-risk group, 76% of stage II and III cancers and 59% of stage 0 cancers were observed (P < 0.01). Using mammographic features generated from DBT screens, our image-based risk prediction model could guide radiologists in selecting women for clinical care, potentially leading to earlier detection and improved prognoses.

PACE4-altCT isoform of proprotein convertase PACE4 as tissue and plasmatic biomarker for prostate cancer.

The proprotein convertase PACE4 has demonstrated value as a viable therapeutic target in prostate cancer (PCa). A novel isoform named PACE4-altCT, which arises in neoplastic lesions, plays an important role in tumor progression and has been validated as a pharmacological target. With the discovery of its overexpression in PCa and the alternative splicing of its pre-RNA to generate an oncogenic C-terminally modified isoform named PACE4-altCT, understanding and validating its value as a potential biomarker is of great interest either from prognostic or targeted therapy intervention. Expression of ERG in LNCaP cells was used to investigate the relationship between ERG expression occurring in PCa cells and PACE4-altCT expression by Western blot and qPCR. Using immunohistochemistry, the expression levels of PACE4 isoforms in patient tissues were investigated and correlated with ERG tumor status and Gleason score. An ELISA method was developed using affinity purified recombinant protein and used for quantitative analysis of plasma concentrations of PACE4-altCT and used for correlation. In contrast with the consensual isoform, PACE4-altCT was only strongly overexpressed in prostate cancer patients, correlated with ERG expression levels. Despite its intracellular retention PACE4-altCT could be detected in the plasma of most patients with prostate cancer, whereas it was only found at low levels in normal patients whereas total plasmatic PACE4 levels did not vary significantly between groups. Our study demonstrates that PACE4-altCT is strongly overexpressed in prostate cancer using both immunohistochemical and ELISA techniques and may have some interesting potential as a biomarker.

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System.

DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus.

APC Mutations Are Not Confined to Hotspot Regions in Early-Onset Colorectal Cancer.

While overall colorectal cancer (CRC) cases have been declining worldwide there has been an increase in the incidence of the disease among patients under 50 years of age. Mutation of the APC gene is a common early event in CRC but is reported at lower rates in early-onset colorectal cancer (EOCRC) than in older patients. Here we investigate the APC mutation status of a cohort of EOCRC patients in New Zealand using a novel sequencing approach targeting regions of the gene encompassing the vast majority of known APC mutations. Using this strategy we find a higher rate (72%) of APC mutation than previously reported in EOCRC with mutations being spread throughout the gene rather than clustered in hotspots as seen with sporadic mutations in older patients. The rate of mutations falling within hotspots was similar to those previously seen in EOCRC and as such our study has implications for sequencing strategies for EOCRC patients. Overall there were low rates of both loss of heterozygosity and microsatellite instability whereas a relatively high rate (40%) of APC promoter methylation was found, possibly reflecting increasing exposure of young people to pro-oncogenic lifestyle factors.

Circulating tumor DNA is a sensitive marker for routine monitoring of treatment response in advanced colorectal cancer.

Accurate assessment of chemotherapy response provides the means to terminate ineffective treatment, trial alternative drug regimens or schedules and reduce dose to minimize toxicity. Here, we have compared circulating tumor DNA (ctDNA) with carcinoembryonic antigen (CEA) for the cycle by cycle assessment of chemotherapy response in 30 patients with metastatic colorectal cancer. CtDNA (quantified using individualized digital droplet PCR (ddPCR) assays) and CEA levels were determined immediately prior to each chemotherapy cycle over time periods ranging from 42-548 days (average of 10 time points/patient). Twenty-nine/thirty (97%) patients had detectable ctDNA compared with 83% whose tumors were CEA-positive (>5 ng/ml) during the monitoring course. Over the course of treatment, 20 disease progression events were detected by computed tomography; ctDNA predicted significantly more of these events than CEA (16 (80%) versus 6 (30%), respectively; P-value = 0.004). When progression was detected by both ctDNA and CEA, the rise in ctDNA occurred significantly earlier than CEA (P-value = 0.046). Partial responses to chemotherapy were also detected more frequently by ctDNA, although this was not significant (P-value = 0.07). In addition, another 28 colorectal cancer patients who underwent potentially curative surgery and showed no evidence of residual disease were monitored with ctDNA for up to 2 years. Clinical relapse was observed in 6/28 (21%) patients. Four out of 6 of these patients showed a significant increase in ctDNA at or prior to relapse. Overall, ctDNA analyses were able to be performed in a clinically relevant timeline and were a more sensitive and responsive measure of tumor burden than CEA.

Estrogen Receptors and Estrogen-Induced Uterine Vasodilation in Pregnancy.

Normal pregnancy is associated with dramatic increases in uterine blood flow to facilitate the bidirectional maternal-fetal exchanges of respiratory gases and to provide sole nutrient support for fetal growth and survival. The mechanism(s) underlying pregnancy-associated uterine vasodilation remain incompletely understood, but this is associated with elevated estrogens, which stimulate specific estrogen receptor (ER)-dependent vasodilator production in the uterine artery (UA). The classical ERs (ERα and ERß) and the plasma-bound G protein-coupled ER (GPR30/GPER) are expressed in UA endothelial cells and smooth muscle cells, mediating the vasodilatory effects of estrogens through genomic and/or nongenomic pathways that are likely epigenetically modified. The activation of these three ERs by estrogens enhances the endothelial production of nitric oxide (NO), which has been shown to play a key role in uterine vasodilation during pregnancy. However, the local blockade of NO biosynthesis only partially attenuates estrogen-induced and pregnancy-associated uterine vasodilation, suggesting that mechanisms other than NO exist to mediate uterine vasodilation. In this review, we summarize the literature on the role of NO in ER-mediated mechanisms controlling estrogen-induced and pregnancy-associated uterine vasodilation and our recent work on a "new" UA vasodilator hydrogen sulfide (H2S) that has dramatically changed our view of how estrogens regulate uterine vasodilation in pregnancy.

Upregulation of PACE4 in prostate cancer is not dependent on E2F transcription factors.

Recent studies in prostate cancer have identified PACE4, a proprotein convertase enzyme, as an emerging therapeutic target. Inhibition of PACE4-altCT, an oncogenic isoform of PACE4, using molecular or pharmacological approaches results in decreased cell proliferation and tumor progression in xenograft models. Although several validations have confirmed PACE4-altCT as a novel therapeutic target, the transcriptional regulation of PACE4 isoforms and mechanism of action remain a challenge. Previously, it has been reported that the human PACE4 promoter possesses potential binding sites for the E2F family of transcription factors, all of which are involved in cell cycle regulation and synthesis of DNA in mammalian cells. Therefore, we attempted to conduct in-depth evaluation of E2Fs on PACE4 and PACE4 isoform expression in prostate cancer. We conducted in vitro molecular silencing studies in various prostate cancer cell lines and determined the change in PACE4 expression levels. The results clearly show that the E2Fs alone do not alter PACE4 expression.

A non-coding genetic variant associated with abdominal aortic aneurysm alters ERG gene regulation.

Abdominal aortic aneurysm (AAA) is a major cause of sudden death in the elderly. While AAA has some overlapping genetic and environmental risk factors with atherosclerosis, there are substantial differences, and AAA-specific medication is lacking. A recent meta-analysis of genome-wide association studies has identified four novel single-nucleotide polymorphisms (SNPs) specifically associated with AAA. Here, we investigated the gene regulatory function for one of four non-coding SNPs associated with AAA, rs2836411, which is located in an intron of the ERG gene. Rs2836411 resides within a >70 kb super-enhancer that has high levels of H3K27ac and H3K4me1 in vascular endothelial and haematopoietic cell types. Enhancer luciferase assays in cell lines showed that the risk allele significantly alters enhancer activity. The risk allele also correlates with reduced ERG expression in aortic and other vascular tissues. To identify whether rs2836411 directly contacts the promoters of ERG and/or of genes further away, we performed allele-specific circular chromosome conformation capture sequencing. In vascular endothelial cells, which express ERG, the SNP region interacts highly within the super-enhancer, while in vascular smooth muscle cells, which do not express ERG, the interactions are distributed across a wider region that includes neighbouring genes. Furthermore, the risk allele has fewer interactions within the super-enhancer compared to the protective allele. In conclusion, our results indicate that rs2836411 likely affects ERG expression by altering enhancer activity and changing local chromatin interactions. ERG is involved in vascular development, angiogenesis, and inflammation in atherosclerosis; therefore mechanistically, rs2836411 could contribute to AAA by modulating ERG levels.

Ser-Phosphorylation of PCSK9 (Proprotein Convertase Subtilisin-Kexin 9) by Fam20C (Family With Sequence Similarity 20, Member C) Kinase Enhances Its Ability to Degrade the LDLR (Low-Density Lipoprotein Receptor).

OBJECTIVE: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. CONCLUSIONS: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.

V-ATPase-associated prorenin receptor is upregulated in prostate cancer after PTEN loss.

Phosphatase and tensin homolog (PTEN) tumor suppressor protein loss is common in prostate cancer (PCa). PTEN loss increases PI3K/Akt signaling, which promotes cell growth and survival. To find secreted biomarkers of PTEN loss, a proteomic screen was used to compare secretomes of cells with and without PTEN expression. We showed that PTEN downregulates Prorenin Receptor (PRR) expression and secretion of soluble Prorenin Receptor (sPRR) in PCa cells and in mouse. PRR is an accessory protein required for assembly of the vacuolar ATPase (V-ATPase) complex. V-ATPase is required for lysosomal acidification, amino acid sensing, efficient mechanistic target of Rapamycin complex 1 (mTORC1) activation, and ß-Catenin signaling. On PCa tissue microarrays, PRR expression displayed a positive correlation with Akt phosphorylation. Moreover, PRR expression was required for proliferation of PCa cells by maintaining V-ATPase function. Further, we provided evidence for a potential clinical role for PRR expression and sPRR concentration in differentiating low from high Gleason grade PCa. Overall, the current study unveils a mechanism by which PTEN can inhibit tumor growth. Lower levels of PRR result in attenuated V-ATPase activity and reduced PCa cell proliferation.

Enhanced anti-tumor activity of the Multi-Leu peptide PACE4 inhibitor transformed into an albumin-bound tumor-targeting prodrug.

The proprotein convertase PACE4 has been validated as a potential target to develop new therapeutic interventions in prostate cancer (PCa). So far, the most effective compound blocking the activity of this enzyme has been designed based on the structure of a small peptide Ac-LLLLRVKR-NH2 known as the Multi-Leu (ML) peptide. Optimization of this scaffold led to the synthesis of compound C23 (Ac-[DLeu]LLLRVK-amidinobenzylamide) with a potent in vivo inhibitory effect on the tumor growth. However, further developments of PACE4 inhibitors may require additional improvements to counter their rapid renal clearance and to increase their tumor targeting efficiency. Herein, we explored the transformation of the ML-peptide into an albumin-binding prodrug containing a tumor specific release mechanism based on the prostate-specific antigen. Our data confirms that intravenous treatment using the ML-peptide alone has little effect on tumor growth, whereas by using the ML-prodrug in LNCaP xenograft-bearing mice it was significantly reduced. Additionally, excellent in vivo stability and tumor-targeting efficiency was demonstrated using a radiolabelled version of this compound. Taken together, these results provide a solid foundation for further development of targeted PACE4 inhibition in PCa.

Germline CDH1 mutations are a significant contributor to the high frequency of early-onset diffuse gastric cancer cases in New Zealand Maori.

New Zealand Maori have a considerably higher incidence of gastric cancer compared to non-Maori, and are one of the few populations worldwide with a higher prevalence of diffuse-type disease. Pathogenic germline CDH1 mutations are causative of hereditary diffuse gastric cancer, a cancer predisposition syndrome primarily characterised by an extreme lifetime risk of developing diffuse gastric cancer. Pathogenic CDH1 mutations are well described in Maori families in New Zealand. However, the contribution of these mutations to the high incidence of gastric cancer is unknown. We have used next-generation sequencing, Sanger sequencing, and Multiplex Ligation-dependent Probe Amplification to examine germline CDH1 in an unselected series of 94 Maori gastric cancer patients and 200 healthy matched controls. Overall, 18% of all cases, 34% of cases diagnosed with diffuse-type gastric cancer, and 67% of cases diagnosed aged less than 45 years carried pathogenic CDH1 mutations. After adjusting for the effect of screening known HDGC families, we estimate that 6% of all advanced gastric cancers and 13% of all advanced diffuse-type gastric cancers would carry germline CDH1 mutations. Our results demonstrate that germline CDH1 mutations are a significant contributor to the high frequency of diffuse gastric cancer in New Zealand Maori.

Improving the Selectivity of PACE4 Inhibitors through Modifications of the P1 Residue.

Paired basic amino acid cleaving enzyme 4 (PACE4), a serine endoprotease of the proprotein convertases family, has been recognized as a promising target for prostate cancer. We previously reported a selective and potent peptide-based inhibitor for PACE4, named the multi-Leu peptide (Ac-LLLLRVKR-NH2 sequence), which was then modified into a more potent and stable compound named C23 with the following structure: Ac-dLeu-LLLRVK-Amba (Amba: 4-amidinobenzylamide). Despite improvements in both in vitro and in vivo profiles of C23, its selectivity for PACE4 over furin was significantly reduced. We examined other Arg-mimetics instead of Amba to regain the lost selectivity. Our results indicated that the replacement of Amba with 5-(aminomethyl)picolinimidamide increased affinity for PACE4 and restored selectivity. Our results also provide a better insight on how structural differences between S1 pockets of PACE4 and furin could be employed in the rational design of selective inhibitors.

Evaluation of PACE4 isoforms as biomarkers in thyroid cancer.

BACKGROUND: To date, no single molecular marker has been demonstrated as clinically useful in differentiating malignant from benign thyroid nodules when a fine needle aspiration falls in the "unknown significance" categories of the Bethesda Classification. PACE4, a member of the proprotein convertase family of enzymes, has been shown to play a major role in the pathogenesis of prostate cancer, through the formation of an oncogenic isoform named PACE4-altCT. PACE4 isoforms have also been suggested to play a role in other cancers, including thyroid cancer, but have never been investigated in a detailed manner. Our objective is to compare the histochemical distribution of the two major PACE4 isoforms in benign and malignant thyroid nodules, in order to determine their potential usefulness as discriminatory biomarkers. METHODS: Thyroid tissues of patients who underwent thyroidectomy were classified according to final pathology. Corresponding tissue sections were immunostained, using two previously validated antibodies raised against the C-terminal end of the two PACE4 isoforms, namely the full-length PACE4 protein (PACE4-FL) and its alternative isoform (PACE4-altCT). Nodules were compared with adjacent normal parenchyma and immunostaining was rated as "low" or "high" by a head and neck pathologist. RESULTS: Non-lesional thyroid parenchyma did not express PACE4-FL (p = 0.002). As a group, malignant (n = 17) nodules expressed PACE4-FL significantly more than benign (n = 24) nodules (percentage of high immunostaining: 52.9% vs 4.2%; p = 0.001). Reciprocally, there was a statistically lower expression of PACE4-altCT in malignant nodules than in adjacent non-lesional parenchyma (p = 0.014). The specificity of a high PACE4-FL immunostaining in determining malignancy was 95.8% (95% CI, 78.9% to 99.9%). CONCLUSION: This study supports the previously described relationship between PACE4-FL and PACE4-altCT through alternative splicing. It also suggests that PACE4-FL is a promising biomarker for thyroid malignancy. Its high specific expression for malignancy could make it an interesting "rule in" test for thyroid cancer. Further prospective, quantitative studies are currently being designed to address how measurements of PACE4 isoforms could be used in a clinical setting. TRIAL REGISTRATION: This study does not report the results of a health care intervention on human participants. It was nonetheless registered on ClinicalTrials.gov under reference number NCT03160482 .

Mouse Mast Cell Protease 4 Deletion Protects Heart Function and Survival After Permanent Myocardial Infarction.

Chymase, a mast cell serine protease involved in the generation of multiple cardiovascular factors, such as angiotensin II and endothelin-1 (ET-1), is elevated and participates in tissue degeneration after permanent myocardial infarction (PMI). Anesthetized 4-month old male wild-type (WT) C57BL/6J mice and mouse mast cell protease-4 knockout (mMCP-4 KO) congeners were subjected to ligation of the left anterior descending (LAD) coronary artery. A group of mice was then subjected to Kaplan-Meier 28-day survival analysis. In another group of mice, 18F-fluorodeoxyglucose positron emission tomography (PET) was performed to evaluate heart function and the infarcted zone 3 days post-PMI surgery. Cardiac morphology following PMI was evaluated on formalin-fixed heart slices and glycoproteomic analysis was performed using mass spectrometry. Finally, cardiac and lung tissue content of immunoreactive ET-1 was determined. PMI caused 60% mortality in WT mice, due to left ventricular wall rupture, and 7% in mMCP-4 KO mice. Cardiac PET analysis revealed a significant reduction in left ventricular volume (systolic and diastolic) and preserved the ejection fraction in mMCP-4 KO compared to WT animals. The infarcted area, apoptotic signaling and wall remodeling were significantly decreased in mMCP-4 KO mice compared to their WT congeners, while collagen deposition was increased. Glycoproteomic analysis showed an increase in apolipoprotein A1, an established chymase substrate in mMCP-4 KO mice compared to WT mice post-PMI. ET-1 levels were increased in the lungs of WT, but not mMCP-4 KO mice, 24 h post-PMI. Thus, the genetic deletion of mMCP-4 improved survival and heart function post-PMI.