Biofilm is a community of bacteria enclosed in an extracellular polymeric substance (EPS) attached to a surface. Inside the biofilm, bacteria can collaborate to increase their survival. The EPS also protects bacteria from drug penetration, leading to increased antibiotic resistance. Therefore, biofilm formation is often linked with chronic bacterial infections. Pseudomonas aeruginosa is an opportunistic pathogen that often causes chronic lung infections in cystic fibrosis patients. It is also a common model for studying biofilm formation. The initial step for biofilm formation is bacteria attaching to and sensing a surface. Upon surface contact, P. aeruginosa may produce cyclic adenosine monophosphate (cAMP), which is a universal second messenger that regulates cellular functions in both eukaryotes and prokaryotes. In P. aeruginosa, cAMP is synthesized by two adenylate cyclases, CyaA & CyaB, and degraded by a cAMP phosphodiesterase, CpdA. cAMP is a key regulator for P. aeruginosa virulence by upregulating the production of the type III secretion system, the type II secretion system, and the type IV pili. However, recent observations in our lab suggest that cAMP may also contribute to the homeostasis of the cell envelope. To investigate this phenomenon, I used microscopy to characterize the cell morphology of strains with different cAMP levels and found that increased cAMP levels lead to longer cells. I also found that high cAMP strains are more sensitive to ꞵ-lactam antibiotics specifically, while low cAMP strains become more resistant. Ongoing work includes characterizing the genetic factors that connect cAMP and ꞵ-lactam sensitivity, as well as using microscopy to determine changes in cell envelop induced by cAMP. Overall, this work reveals an important role of cAMP in bacterial physiology and provides insight into the complex relationship between virulence and antimicrobial resistance.

Engineered heart tissues (EHTs) have emerged as a promising tool for cardiac disease modeling and drug screening, allowing for better study of cardiovascular diseases (CVDs). However, most current EHTs are composed of only a mixture of an extracellular matrix and heart muscle cells, called cardiomyocytes (CMs), without a vascular element. This prevents the study of the impacts of flow and the endothelium on cardiac function, and the role that endothelial cell (EC) dysfunction may play in cardiovascular disease. Endothelial function is closely related to cardiac homeostasis, as risk factors for CVD (smoking, obesity, diabetes, etc.) lead to an increase in pro-inflammatory cytokines, which can trigger EC dysfunction. Thus, this interaction is important to study further. The Zheng lab has developed a perfusable collagen-based EHT model, which incorporates a vascular element. The constructs form a lumen through utilization of needles and collagen, support CMs within the bulk collagen matrix, and the inner lumen of the tube can be endothelialized, serving as an effective in vitro model of cardiac vasculature. This project aims to identify healthy and unhealthy EC flow conditions within the EHTs, hypothesizing that physiologically relevant shear stress will lead to EC alignment and strong barrier properties. . We optimized the fabrication and culture process of the EHTs by fabricating a secondary dish for the EHT constructs while they are under perfusion, in order to avoid contamination risks. We then employed this model to look at EC retention and health at different flow rates, and examined the effects of altered shear stress on EC dysfunction. ECs perfused under physiological shear stress have shown markers of healthy barrier function and alignment. This project establishes a perfusable EHT model that allows us to interrogate EC function under perfusion and, in the future, assess the effect of endothelial dysfunction on cardiac dysfunction.

Pulmonary Arterial Hypertension (PAH) is a deadly vascular disease, affecting the blood vessels of the lungs rather than the systemic circulation, with no existing cure. PAH is characterized by pulmonary arterial smooth muscle cell (PASMC) hypertrophy and hyperplasia, which increases resistance to blood flow within the pulmonary arteries and leads to rapid symptom progression and death from right heart failure over several years. We hypothesize that defects in PASMC differentiation and alignment may contribute to PAH. Prior work has shown that micropatterned scaffolds encourage vascular SMC alignment and differentiation towards a contractile phenotype. To test whether these responses differ in patients with PAH, we designed a micropatterned collagen scaffold atop a glass coverslip. Scaffoldings were imprinted with either alternating 10-µm wide x 10-µm deep microchannels or left unpatterned. Explanted PASMCs from patients with PAH or failed donors (controls) were cultured on patterned versus unpatterned constructs and alignment, protein expression, and cellular morphology were compared across conditions. I evaluated 3 PAH and 3 control subjects and have collected preliminary data for each condition (control versus PAH), with three technical replicates each. Through these preliminary studies, I have demonstrated success of my model with consistent alignment observed on patterned substrates. Excitingly, PASMCs from patients with PAH expressed significantly decreased levels of the contractile protein, Calponin, when compared with control cells, including after responding to cues that promote alignment and contractility. This suggests that PAH PASMCs remain in an inappropriately synthetic or proliferative state. Subsequent testing will include assessment of calcium signaling in response to contractile stimuli and transcriptomic evaluation of cellular responses to micropatterning. This work will enhance understanding of whether SMC abnormalities contribute to disease initiation and progression in PAH and will contribute to the broader effort of developing more complex models of pulmonary vascular disease.