Myocardial infarction (MI) is the leading cause of death in patients suffering from cardiovascular disease. MI, which limits blood and oxygen to the myocardial tissue, causes the loss of contractile cardiomyocytes, which are replaced by activated cardiac fibroblasts (cFBs) that express extracellular matrix (ECM) proteins comprising fibrotic tissue. We recently discovered that collagen type XVIII alpha 1 chain (Col18a1) is particularly enriched in cFBs in the heart and that Col18a1 expression is increased in activated cFBs found within the fibrotic tissue after MI. Endostatin, a C-terminal protein fragment derived from Col18a1, is upregulated in the serum and/or tissue of experimental cardiac disease models and human patients after MI, but its role in activated cFBs is unknown. To investigate the effect of endostatin on the activation of cFBs, we purified adult cFBs from C57BL/6 mice for in vitro studies. Activation of cFBs was induced by incubating cells with transforming growth factor-b (TGFb) and Angiotensin II (AngII), followed by endostatin or vehicle treatments. After 48 hours, cells were lysed for RNA isolation, cDNA synthesis, and gene expression evaluation of fibrotic genes using qRT-PCR. Here, we found that expression of ECM genes (Postn, Fn, Col1a2) was increased in cFBs treated with endostatin compared to TGFb/AngII alone. Future extensions include reinforcement of these findings within in vivo MI models. By over-expressing endostatin and investigating its influence on other genes involved within fibrosis, we hope to understand better its role in cardiac remodeling to develop a treatment with the potential to help millions.

Our laboratory has discovered that histone H3 is an oxidoreductase enzyme that reduces Cu2+ into Cu1+ (Attar et. al. 2020). The enzymatic activity of histone H3 is therefore important to maintain copper in its biousable reduced state (Cu1+). Maintaining copper homeostasis is essential to cell health, as too little is insufficient for proper cellular function but too much can cause mis-metalation of proteins, protein aggregation, and oxidative damage (Chen et. al., 2022; Zuily et. al. 2022). This motivated our investigation into cellular adaptations in response to a weakened enzymatic activity of histone H3. In previous studies with Saccharomyces Cerevisiae, the region in the ribosomal DNA locus (rDNA), becomes active in a strain with decreased histone copper reductase activity (H3H113N). This suggested that there may be a relationship between Sir2-mediated control of rDNA locus transcription and copper reduction by histone H3. To investigate this, we deleted the SIR2 gene in wildtype (WT) yeast as well as strains with a loss (H3H113N) or gain of function (H3A110C) mutation in histone H3. We used growth assays and a fluorescent-based intracellular Cu1+ reporter to characterize the strains. We found that while sir2Δ in WT and H3A110C had little effect on growth, it improved the defective growth of H3H113N in oxidative media and substantially enhanced its resistance to copper toxicity. These findings point to a relationship between histone copper reductase activity, intracellular copper oxidation state and Sir2. Future experiments will be aimed at understanding the molecular basis of this relationship.

Numerous diseases show sex bias, such as autoimmunity in which females make up 80% of the patient population. Females and males differ in sex hormones and chromosome complement (generally XX or XY), but epidemiologic and animal studies provide evidence of X-chromosome effects independent of hormonal influence. Many X-linked genes are linked to immune function, and females have 2 copies of these genes. In order to equalize expression across sexes, one of a female's 2 X chromosomes is inactivated through a process called X-chromosome inactivation. Despite this, 15-20% of genes escape inactivation and are expressed biallelically. Escape of immune-related genes can lead to an overactive immune response in females. However, escape varies by gene and context. For instance, Oghumu et. al showed that L. mexicana infection caused increased CXCR3 escape. Therefore, it is important to uncover which conditions can cause which genes to escape. Thus, we looked to see if FOXP3, a known X-linked suppressive immune regulatory gene, escapes inactivation under homeostatic conditions or autoimmune colitis. We hypothesized that if mice are presented with an inflammatory disease, a suppressive immune gene (i.e. FOXP3) will escape inactivation because a less active immune system would be beneficial for the individual. We did not observe FOXP3 escape under either condition. Moving forward, we will explore whether epigenetic mechanisms may play a role in keeping FOXP3 inactivated. Overall, this project increases our understanding of the complexity of X-chromosome inactivation escape and points towards novel therapies that could manipulate X-linked gene expression in immune disease.

Placenta Accreta Spectrum (PAS) is linked to severe maternal morbidity and mortality, with its incidence rising due to increased cesarean births globally, including in the United States. Conventionally viewed as a trophoblast-related disorder, recent literature has shifted focus towards flawed decidua and uterine dehiscence. The extracellular matrix underlying PAS has not been described. This study aims to characterize collagen distribution within PAS-afflicted placentas, investigating potential discrepancies based on placental adherence patterns in PAS. Unstained fixed biopsies from cesarean hysterectomies were analyzed to observe collagen distribution and subtypes. Participants were recruited, and written informed consent was obtained before delivery. The study employed label-free multiphoton microscopy techniques: Second Harmonic Generation (SHG) for collagen types 1 & 2 and Fluorescence Lifetime Imaging Microscopy (FLIM) to isolate collagen types 1 and 3 using phasor-based lifetime analysis. Within the same placenta in patients with PAS, there is an increase in type 1 collagen at the site of most placental adherence. Additionally, type 1 collagen is attenuated in the placenta, which is non-adherent with a tropism to the uterine endometrium and myometrium. An increase in collagen at the site of most commitment in PAS supports the theory that PAS is related to a loss of regular barrier function at the uterine-placental interface. By revealing the structure and subtypes of collagen at the fetal-maternal interface, we aim to understand the underlying mechanism of PAS via a detailed description of the collagen profile in PAS.