Lysyl oxidases (LOXs), comprising lysyl oxidase (LOX) and lysyl oxidase like 1-4 (LOXL1-4), constitute a highly conserved enzyme family. Their primary function is the crosslinking of collagen and elastic fibers, thereby modulating the structure and function of the extracellular matrix. Our primary objective was to elucidate the localization of LOXs within human endometrial tissues and to investigate the expression profile of LOXs during in vitro human endometrial stromal cell decidualization. Immunohistochemical localization revealed that all LOXs were expressed in human endometrium during both the proliferative and secretory phases of menstrual cycle. All five LOXs were expressed in human endometrial stromal cells cultured in vitro . Gene expression levels of LOX, LOXL1, and LOXL2 significantly decreased during endometrial stromal cell decidualization in vitro . In contrast, LOXL3 and LOXL4 gene expression remained unaffected. Protein levels of LOX, LOXL1, and LOXL3 also showed a significant reduction. Additionally, conditioned media analysis revealed abundant secretion and release of LOX and LOXL1-3 into the extracellular space at all time points examined, with their levels remaining constant. The primary targets of LOXs, collagen and elastic fibers, undergo significant synthesis and processing during endometrial stromal cell decidualization in vitro . This observation was supported by the differential gene expression levels of factors involved in their processing and assembly. Collectively, this study demonstrates the expression of LOXs in the human endometrium and their potential role in extracellular matrix reorganization during decidualization. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Although many studies suggested the benefit of smoking cessation among pregnant women in reducing the risk of preterm birth (PTB), the timing of the effect of the cessation remains inconclusive. OBJECTIVES:To examine the association of trimester-specific smoking cessation behaviours with PTB risk. METHODS:We included 199,453 live births in Western New York between 2004 and 2018. Based on self-reported cigarette smoking during preconception and in each trimester, we created six mutually exclusive groups: non-smokers, quitters in each trimester, those who smoked throughout pregnancy, and inconsistent smokers. Risk ratios (RRs) and 95% confidence intervals (CIs) were estimated using Poisson regression to examine the association between smoking cessation and PTB. Effect modification by illegal drug use, maternal age, race and ethnicity and pre-pregnancy body mass index (BMI) was investigated multiplicatively by ratio of relative risk and additively by relative excess risk due to interaction (RERI). RESULTS:Overall, 6.7% of women had a PTB; 14.1% smoked throughout pregnancy and 3.4%, 1.8% and 0.8% reported quitting smoking during the first, second and third trimesters, respectively. Compared to non-smokers, third-trimester cessation (RR 1.20, 95% CI 1.01, 1.43) and smoking throughout pregnancy (RR 1.27, 95% CI 1.21, 1.33) were associated with a higher PTB risk, while quitting smoking during the first or second trimester, or inconsistent smoking was not associated with PTB. A positive additive interaction was identified for maternal age and late smoking cessation or smoking throughout pregnancy on PTB risk (RERI 0.17, 95% CI 0.00, 0.36), and a negative interaction was observed for pre-pregnancy BMI ≥30 kg/m2 (ratio of relative risk 0.70, 95% CI 0.63, 0.78; RERI -0.42, 95% CI -0.56, -0.30). CONCLUSION:Compared to non-smokers, smoking throughout pregnancy and third-trimester smoking cessation are associated with an increased risk of PTB, while quitting before the third trimester may not increase PTB risk.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Children with beta-thalassemia (BT) present with an increase in carotid intima-medial thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. To evaluate this, wild-type (WT, littermates) and BT (Hbbth3/+) mice, both male and female, were placed on a 3-mo high-fat diet with low-density lipoprotein receptor suppression via overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation (D377Y). Mechanistically, we hypothesize that heme-mediated oxidative stress creates a proatherogenic environment in BT because BT is a hemolytic anemia that has increased free heme and exhausted hemopexin, heme's endogenous scavenger, in the vasculature. We evaluated the effect of hemopexin (HPX) therapy, mediated via an adeno-associated virus, to the progression of atherosclerosis in BT and a phenylhydrazine-induced model of intravascular hemolysis. In addition, we evaluated the effect of deferiprone (DFP)-mediated iron chelation in the progression of atherosclerosis in BT mice. Aortic en face and aortic root lesion area analysis revealed elevated plaque accumulation in both male and female BT mice compared with WT mice. Hemopexin therapy was able to decrease plaque accumulation in both BT mice and mice on our phenylhydrazine (PHZ)-induced model of hemolysis. DFP decreased atherosclerosis in BT mice but did not provide an additive benefit to HPX therapy. Our data demonstrate for the first time that the underlying pathophysiology of BT leads to accelerated atherosclerosis and shows that heme contributes to atherosclerotic plaque development in BT.NEW & NOTEWORTHY This work definitively shows for the first time that beta-thalassemia leads to accelerated atherosclerosis. We demonstrated that intravascular hemolysis is a prominent feature in beta-thalassemia and the resulting increases in free heme are mechanistically relevant. Adeno-associated virus (AAV)-hemopexin therapy led to decreased free heme and atherosclerotic plaque area in both beta-thalassemia and phenylhydrazine-treated mice. Deferiprone-mediated iron chelation led to deceased plaque accumulation in beta-thalassemia mice but provided no additive benefit to hemopexin therapy.

Pelvic pain in women with endometriosis is attributed to neuroinflammation and afferent nociceptor nerves in ectopic and eutopic endometrium. The hypothesis that uterine nociception is activated by IL-1β, a prominent cytokine in endometriosis, was tested herein. Immunofluorescence histochemistry confirmed the presence of neurons in human endometrial tissue. Expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and their receptors in endometrial tissue and cells was validated by immunohistochemistry and Western blotting. Isolated endometrial stromal cells (ESCs) were subjected to dose-response and time-course experiments with IL-1β and kinase inhibitors to characterize in vitro biomarkers. Neural biomarkers were co-localized in endometrial nerve fibers. NGF, BDNF, and their receptors tropomyosin receptor kinase (Trk) A, TrkB, and p75 neurotrophin receptor were all expressed in primary ESCs. IL-1β stimulated higher TrkA/B expression in ESCs derived from endometriosis cases (2.8- ± 0.2-fold) than cells from controls (1.5- ± 0.3-fold, t-test, P < 0.01), effects that were mediated via the c-Jun N-terminal kinase (JNK) pathway. BDNF concentrations trended higher in peritoneal fluid of endometriosis cases but were not statistically different from controls (P = 0.16). The results support the hypothesis that NGF and BDNF and their corresponding receptors orchestrate innervation of the endometrium, which is augmented by IL-1β. We postulate that JNK inhibitors, such as SP600125, have the potential to reduce neuroinflammation in women with endometriosis.

Abstract Endometriosis is a common gynecological inflammatory disorder, which is characterized by immune system dysregulation with initiation and progression. It affects 5% to 15% of reproductive-age women and is present in as many as 30% to 50% of patients with infertility and/or pain. In previous studies, including ours, have demonstrated that several cytokines have been associated with the evolution of endometriosis, including tumor necrosis factor-a (TNFa). TNFa is a non-glycosylated protein which has potent inflammatory, cytotoxic, and angiogenic potential. Therefore, in the current studies, we examined the effects of TNFa over a time course in the regulation of proinflammatory and proangiogenic microRNAs (miRNAs) in primary cultures of normal endometrial stromal cells (NESC) and compared with the untreated cells derived from eutopic endometrium of endometriosis subjects (EESC). miRNAs are short, 18- to 22-nucleotide– size, non-coding RNAs that act as post-transcriptional modulators of gene expression and are involved in the pathogenesis of endometriosis. Using NanoString nCounter-based assays and quantitative RT-PCR, we have identified levels of several proinflammatory and proangiogenic miRNAs higher in EESC than NESC. NESC treatment with TNFa significantly altered the expression of proinflammatory and proangiogenic miRNAs in a time-dependent manner. Notably, TNFa significantly decreased phosphorylation of the PI3K, AKT, and ERK signaling pathways. Moreover, treatment of EESC and NESC with curcumin (diferuloylmethane, CUR), an anti-inflammatory folk medicine in Asian countries, significantly increased the expression of anti-inflammatory and anti-angiogenic miRNAs in a dose- and time-dependent manner. These findings demonstrate higher inflammatory, and proangiogenic miRNA production in EESC may be due to a higher concentration of TNFa than NESC under basal conditions. Therefore, suppressing TNFa may reduce the inflammatory and angiogenic miRNA associated with endometriosis. Sources of Research Support: This study was supported in part by National Institutes of Health Grants 1SC3 GM113751, U01, 1SC1 GM130544, HD66439, 1R01HD057235, U54 CA118948, HD41749, S21MD000101 and G12-MD007602. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR018386 from NIH/NCRR. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:06 p.m. - 1:11 p.m.

In the early 1980s, physicians and scientists began to gain an appreciation of the physiological importance of the endothelium, the simple unicellular layer lining the luminal surface of blood vessels. Indeed, in Chesley's first, single-authored edition of this text, the reference to this term was confined to the “endotheliosis” lesion of the renal glomerulus. We now recognize that endothelial cells are critical sensors of the milieu interieur and potent regulators of vascular tone, organ perfusion, and ischemia. The “endothelial hypothesis” of preeclampsia etiology provides for a convergence of several factors thought to play fundamental roles in its pathogenesis: leukocytes, platelets, cytokines, fatty acids, oxygen free radicals, placental microvesicles, cell-free DNA fragments, “antiangiogenic” factors, and autoantibodies are all considered.

This introductory Chapter is devoted to the recognition of Leon Chesley’s immense impact on the study of preeclampsia and hypertensive diseases in pregnancy. In the 43 years following his epic first edition, major advances and giant strides in research have led to new insights regarding the pathophysiology of preeclampsia. Cutting-edge tools in aspects of genetics, cell imaging, immunology, molecular biology, -omics and bioinformatics have facilitated these findings. These advances, particularly in the basic sciences, are a true reflection of the influence of Leon Chesley, a PhD who did not practice medicine. However, he espoused and established the translation of scientific observations to the clinic, hospital, labor and delivery unit, and importantly, to long-term follow-up of women found to be hypertensive during pregnancy. His legacy, application of keen scientific knowledge to improve the practice of clinical obstetrics, is what Dr. Chesley so well communicated in his words and deeds. In this chapter we reproduce Dr. Chesley’s original chapter entitled “History” in its entirety (with minor edits for clarity) and conclude by republishing Dr. Chesley’s 1975 workshop banquet address titled “False Steps in the Study of Preeclampsia.” That workshop begot the establishment of the International Society for the Study of Hypertension in Pregnancy in 1976, an organization that remains committed “to stimulate research in the field of hypertension in pregnancy, disseminate the useful results of such research, and advance education in the field.” Dr. Chesley’s wisdom about how to study preeclampsia remains entirely valid today.

We developed multiple microfluidic organ-on-chip (OOC) devices that represent the structure, functions, and responses of the two feto-maternal interfaces (FMis) in humans (fetal membrane [FMi-OOC] and placenta [PLA-OOC]). Generated by BioRender.

The complex relationships among platelet function, coagulation, and hepatic pathophysiology in pregnancy were recognized since the time of Leon Chesley's first edition of this textbook. Indeed, many of the clinical biochemistry analytes that we rely upon to diagnose and monitor the course of preeclampsia derive from these systems. Further, the combination of microangiopathic hemolysis, hepatocellular disruption, and thrombocytopenia specifies the HELLP syndrome, a critical preeclampsia variant, which is covered in detail in this chapter. The authors review the use of aspirin to prevent pre-eclampsia and steroids for HELLP Syndrome.

In this chapter we have turned to the outstanding expertise of Graham Burton and his colleagues at the Physiological Laboratory at Cambridge. Here the reader will find a detailed description of the microanatomical development of the fetal–maternal interface, particularly as it relates to the biomechanics of the growing placental vasculature. Clinical correlations with high-resolution radiological imaging findings are included. The critical roles of oxygen tension, cell stress, and senescence also are highlighted. Drawing from these experts in the field, students of preeclampsia will be able to appreciate the integrated roles of form and function in the placenta’s normal physiology and under disease states.

Introduction: It is well described that changes in the aortic media are crucial in the development of Abdominal Aortic Aneurysms (AAA), however the involvement of the endothelium is not as clear. The endothelium is the first vascular layer exposed to blood flow and aneurysms preferentially form in areas of disturbed flow. The regulatory mechanisms as to why aneurysms form at these sites remains unclear. Here we propose that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pivotal role in the endothelium’s response to AAA formation in a shear responsive manner. Methods: 10-12 week old male mice deficient in endothelial specific PGC1α (KO) and their corresponding wild type background mice (WT) were treated with 0.75 mg/kg/day of Ang II. Our primary endpoints were measurement of the maximal width of the suprarenal aortas, frequency, and complexity based on the Daugherty classification. To determine the effects of shear, HAECs exposed to steady flow (15 dyn/cm 2 ) and disturbed flow (0±6 dyn/cm 2 at 1 Hz) for 12 hours were compared to determine if mRNA expression were different for PGC1α and SIRT1. Results: AAA incidence and severity were increased in ANG II-infused KO compared to the WT mice (100% vs. 20%, respectively). Measurements of excised aortas showed that KO mice exhibited larger AAA fold-increase compared to WT (2.00 ± 0.11 fold increase vs. 0.43 ± 0.19 fold increase, p-value 0.0002). The KO male mice also exhibited a more complex phenotype. HAECs that underwent disturbed flow had a significant decrease in expression of SIRT1 (p = 0.02) and trended towards a decrease in expression of PGC1α (p = 0.06). Conclusions: Our data strongly suggests that knockout of endothelial specific PGC1α leads to more frequent, complex and larger AAAs primarily in the suprarenal aorta, which is an area of disturbed flow. Interestingly, the addition of ANG II alone was able to induce AAA formation in our model. Additionally, we show that PGC1α and SIRT1 are regulated in a shear responsive manner. Taken together, endothelial PGC1α likely plays a regulatory role in AAA formation through shear-related mechanisms.

Rationale: Doxorubicin is a widely used anticancer drug. However, its major side effect, cardiotoxicity, results from cardiomyocyte loss that causes left ventricle (LV) wall thinning, chronic LV dysfunction and heart failure. Cardiomyocyte number expansion by thyroid hormone (T3) during preadolescence is suppressed by the developmental induction of an ERK1/2-specific dual specificity phosphatase 5 (DUSP5). Here, we sought to determine if a brief course of combined DUSP5 suppression plus T3 therapy replaces cardiomyocytes lost due to preexisting doxorubicin injury and reverses heart failure. Methods: We used in vivo-jetPEI to deliver DUSP5 or scrambled siRNA to ~5-week-old C57BL6 mice followed by 5 daily injections of T3 (2 ng/µg body weight). Genetic lineage tracing using Myh6-MerCreMer::Rosa26fs-Confetti mice and direct cardiomyocyte number counting, along with cell cycle inhibition (danusertib), was used to test if this treatment leads to de novo cardiomyocyte generation and improves LV contractile function. Three doses of doxorubicin (20 µg/g) given at 2-weekly intervals, starting at 5-weeks of age in C57BL6 mice, caused severe heart failure, as evident by a decrease in LV ejection fraction. Mice with an ~40 percentage point decrease in LVEF post-doxorubicin injury were randomized to receive either DUSP5 siRNA plus T3, or scrambled siRNA plus vehicle for T3. Age-matched mice without doxorubicin injury served as controls. Results: In uninjured adult mice, transient therapy with DUSP5 siRNA and T3 increases cardiomyocyte numbers, which is required for the associated increase in LV contractile function, since both are blocked by danusertib. In mice with chronic doxorubicin injury, DUSP5 siRNA plus T3 therapy rebuilds LV muscle by increasing cardiomyocyte numbers, which reverses LV dysfunction and prevents progressive chamber dilatation. Conclusion: RNA therapies are showing great potential. Importantly, a GMP compliant in vivo-jetPEI system for delivery of siRNA is already in use in humans, as is T3. Given these considerations, our findings provide a potentially highly translatable strategy for addressing doxorubicin cardiomyopathy, a currently untreatable condition.

Infectious endocarditis is a life-threatening disease, and diagnostics are urgently needed to accurately diagnose this disease especially in the case of prosthetic valve endocarditis. We show here that maltohexaose conjugated to indocyanine green (MH-ICG) can detect Staphylococcus aureus ( S . aureus ) infection in a rat model of infective endocarditis. The affinity of MH-ICG to S . aureus was determined and had a Km and Vmax of 5.4 μM and 3.0 X 10 −6 μmol/minutes/10 8 CFU, respectively. MH-ICG had no detectable toxicity to mammalian cells at concentrations as high as 100 μM. The in vivo efficiency of MH-ICG in rats was evaluated using a right heart endocarditis model, and the accumulation of MH-ICG in the bacterial vegetations was 2.5 ± 0.2 times higher than that in the control left ventricular wall. The biological half-life of MH-ICG in healthy rats was 14.0 ± 1.3 minutes, and approximately 50% of injected MH-ICG was excreted into the feces after 24 hours. These data demonstrate that MH-ICG was internalized by bacteria with high specificity and that MH-ICG specifically accumulated in bacterial vegetations in a rat model of endocarditis. These results demonstrate the potential efficacy of this agent in the detection of infective endocarditis.