Browsing by Subject "Statins"

Advances in tissue engineering have led to the development of 3D biomimetic models of human skeletal muscle (myobundles), which have a wide range of applications in regenerative medicine, predictive drug testing and toxicology screening, and development of therapeutics for muscular disorders. Statins are the most commonly used medications to lower cholesterol and prevent cardiovascular diseases. Long term treatment using statins may lead to musculoskeletal side effects in the form of myopathy, myalgia or rhabdomyolysis. Currently, there are no reliable diagnostic measures of statin-induced myopathy since only some of the individuals complaining of the symptoms of statin myopathy exhibit elevated creatine kinase levels. This study examined if the engineered human skeletal myobundles using cells derived from patients with a history of statin induced myopathy exhibit statin-dependent defects in muscle physiology when exposed to varying concentrations of statins in vitro. Utilizing a microphysiological system for skeletal muscle, that uses patient-derived tissue to form engineered myobundles, to recapitulate the organization and function of native muscle is a novel way to understand the functional changes within individuals that experience statin induced myopathy. To create myobundles, myoblasts were encapsulated in a matrigel/fibrin matrix. The myobundles were cultured in 3D human growth media for 4 days and shifted to low amino acid differentiation media for another 4 days and then dosed for 5 days with varying concentrations of statins in low amino acid differentiation media. These were then force tested to see if there is a difference in twitch, tetanus and fatigue force response between different statin types, concentration levels, case and donor and dose exposure periods. A repeated measures multiple linear regression of the raw tetanus force versus statin dose, myopathy or control, and type of statin revealed a negative relationship between tetanus force response and statin concentration and myopathy. It was also seen that force production for the myopathy donors dosed with simvastatin when compared to their control pair was much lower which can be due to muscle weakness in these donors. In addition, immunofluorescence of the myobundles showed there is a structural difference in myotubule formation between the myopathy donors and control donors which can be correlated to force production. The donors that exhibited fewer myotubules or those that had a frayed appearance and lacked structural definition had lower force production. Overall, myobundles prepared with myoblasts from the myopathy and control donors exhibited functional changes associated with reported statin myopathy, while absence of myofibers or degradation of myofibers was associated with very low levels of force production.

Endothelial dysfunction is the predominant pathophysiological state prior to the onset of atherosclerosis. Currently, treatments for endothelial dysfunction are evaluated in vitro using two-dimensional (2D) cell culture assays or in vivo animal models. Microphysiological systems are small-scale three-dimensional (3D) tissue models that recapitulate the native tissue structure and function. An ideal microphysiological system is comprised of human cells embedded within a 3D matrix introduced to physiological fluid perfusion. Immune challenge in the form of cytokines or immune cells further recapitulates the native microenvironment.

A vascular microphysiological system was developed from a small-diameter tissue engineered blood vessel (TEBV) in a perfusion culture circuit. TEBVs were created from collagen gels embedded with human neonatal dermal fibroblasts and plastically compressed to yield collagen constructs with high fiber densities. TEBVs are rapidly producible and can be directly introduced into perfusion culture immediately after fabrication. Endothelium-independent vasoconstriction in response to phenylephrine and endothelium-dependent vasodilation in response to acetylcholine were used to analyze the health and function of the endothelium non-destructively over time.

Endothelial dysfunction was induced through introduction of the pro-inflammatory cytokine tumor necrosis factor – α (TNF-α). Late-outgrowth endothelial progenitor cells derived from the peripheral blood of coronary artery disease patients (CAD EPCs) were evaluated as a potential endothelial source for autologous implantation in both a two-dimensional (2D) direct co-culture model as well as a 3D model as an endothelial source for a tissue engineered blood vessel. CAD EPCs demonstrated similar adhesive properties to a confluent, quiescent layer of smooth muscle compared to human aortic endothelial cells. Within the TEBV system, CAD EPCs demonstrated the capacity to elicit endothelium-dependent vasodilation. CAD EPCs were compared to adult EPCs from young, healthy volunteers. Both CAD EPCs and healthy volunteer EPCs demonstrated similar endothelium-dependent vasoactivity in response to acetylcholine; however, in response to TNF-α, CAD EPCs demonstrated a reduced response to phenylephrine at high doses.

The treatment of TEBVs with statins was explored to model the drug response within the system. TEBVs were treated with lovastatin, atorvastatin, and rosuvastatin for three days prior to exposure to TNF-α. In all three cases, statins prevented TNF-α induced vasoconstriction in response to acetylcholine within the TEBVs, compared to TEBVs not treated with statins. Overall, this work characterizes and validates a novel vascular microphysiological system that can be tested in situ in order to determine the effects of various patient populations and drugs on endothelial health and function under healthy and inflammatory conditions.

In recent years, our understanding of the scope and diversity of protein post-translational modifications has rapidly expanded. While mitochondrial proteins in particular have been studied in detail and are decorated with an array of acyl groups that can occur non-enzymatically, cytosolic proteins are also known to be acylated. Interestingly, these modifying chemical moieties are often associated with intermediary metabolites from core metabolic pathways. We looked to explore the emerging links between the intrinsic reactivity of metabolites, non-enzymatic protein acylation, and possible signaling roles for these metabolites. Previously HMG-CoA was identified as a reactive metabolite that non-enzymatically modifies proteins when its utilization is blocked. We investigated whether statins, which inhibit the HMG-CoA consuming enzyme HMG-CoA reductase (HMGCR), would result in HMGylation as well. This is particularly relevant because statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Using mass spectrometry based metabolomics, we found that in both cells and mice treated with statins, HMG-CoA levels increased. Surprisingly, this increase in HMG-CoA led to a single protein being HMGylated, which is in contrast to prior work showing a multitude of protein modifications occurring when acyl-CoA consuming enzymes were altered. Using mass spectrometry, we identified the modified protein as fatty acid synthase (FAS), which implied a new connection between two lipid biosynthetic pathways. We thoroughly characterized the chemical nature of the modification through in vitro chemical treatments. These investigations revealed the modification on FAS to be labile and dynamic compared to previously described acyl modifications. The modification was susceptible to heating, reducing agents, as well as displacement by other acyl-CoAs. Additionally, the modification was rapidly induced and removed in cells in response to the addition or removal of statin. This pointed to a modification that was not lysine bound, which is the commonly reported acyl modification, and is very stable. We developed mass spectrometry methods that would stabilize the modification on FAS and were able to locate the site of HMGylation. The modification occurs on both the serine residue responsible for accepting acyl groups, as well as on the prosthetic group that facilitates the growing of the fatty acyl chain. These modifications occur on active site residues and when tested using purified HMGylated FAS, we found the activity to be inhibited in proportion with the amount of HMG-CoA present. We then tested the impact of statin-treatment on the production of fatty acids in both cells and mice. Surprisingly, we found no change in the lipogenesis rates during statin-treatment, despite the in vitro evidence indicating FAS is inhibited when HMGylated. Recent discoveries have shown a role for FAS outside of the canonical production of lipids for energy storage. With the knowledge that there are subpools of FAS localized within the cell, some of which perform signaling functions, we predicted that the HMGylation occurred on a subpool of FAS in close proximity to HMGCR and endoplasmic reticulum (ER) membrane and conveyed a signal. Utilizing cell fluorescence microscopy, we confirmed the interaction of FAS and HMGCR. To look for any possible changes in cellular signaling we utilized label-free quantitative proteomics and identified pathways changing due to the HMGylation of FAS. While the specific mechanism and nature of these changes remains to be discovered, we identified several signatures of ER and Golgi pathways changing in response to FAS HMGylation and discuss possible areas of interest for future investigation. Finally, we used this new information to begin an investigation into broader non-lysine linked modifications. By artificially inducing acylation in cells, we show that heat or reducing agents play a large role in the detection of acyl-PTMs by Western blotting across HMGylation, glutarylation, and acetylation. In summary, we show how the inhibition of a single enzyme can result in the targeted modification of a protein in an adjacent pathway. This modification occurs on a serine residue and prosthetic group, allowing for dynamic modification and regulation of the enzyme. Furthermore, the discovery of these non-lysine residues reveals the possibility for widespread non-lysine acyl PTMs, waiting to be discovered following the revision of current standard methodology.

Background: Hyperlipidemia is increasingly prevalent in China. Gaps are found between 2016 Chinese guideline for lipid management and other international guidelines. This study aims to identify attitudes and reported practice patterns for hyperlipidemia among Chinese physicians in tertiary hospitals. Methods: We collected data for 309 physicians on their adoption of guidelines, their attitudes of statin therapy and reported statin prescription patterns in four hypothetical patient scenarios (low risk/high LDL, high risk/high LDL, low risk/low LDL, and high risk/low LDL patients). Results: Overall, 63.75% of physicians adopted 2016 Chinese guideline. Most highly agreed with statins’ effectiveness, but 57.94% concerned about the safety of high-intensity statins in the Chinese population. Physicians reported various LDL-C value for treatment target. In hypothetical scenarios, the prescription rate was highest for the high risk/high LDL patient (90.03%). Those who believed statins could prevent stroke and heart attack were more likely to prescribe statins (OR=5.67,p=0.002). The prescription rate was 81.42% for the patient at risk/low LDL. Those who believed statins could prolong life were more likely to prescribe (OR=2.51, P=0.009). Only 7.78% 2016 Chinese guideline adopters prescribed statins as guideline recommended on all four hypothetical patients. Most physicians (56.73%-73.91%) preferred moderate-intensity statins. Those who considered high-intensity statins shouldn’t be routinely used in Chinese were less likely to prescribe high-intensity statins(OR=0.33, p=0.004). Conclusions: Physicians concerned about statins’ safety; We didn’t find a specific practice pattern among physicians and guideline adopters’ reported practices were not always concordant with the recommendations; Future studies are expected to focus on high-intensity statins and LDL-C target for treatment and training on guideline use is necessary;