The current work focused on AVHSE as a research tool, but similar techniques could be further developed for the development of grafts. Grafting would require addressing many of the structural and biological limitations noted above, as well as modifications to address host immunity issues. Overall, we have demonstrated AVHSEs as a research platform with regards to photoaging effects, but expansions of this model could be utilized for clinical skin substitutes, personalized medicine, screening of chemicals/cosmetics, drug discovery, wound healing studies, and therapeutic studies. Perfluoroalkyl substances are stable, water-soluble compounds that persist in the environment and are of major concern for the public. These substances are widely used in surfactants, lubricants, paints, polishes, paper/textile coatings, food packaging, and fire retardant . The most widely studied PFAS chemicals are PFOS and PFOA. These are organic compounds in which all hydrogens on all non-functional group associated carbons have been replaced by fluorine; thus, PFOS and PFOA are extremely stable due to their numerous strong carbon-fluorine bonds100 . Due to their stability and bio-accumulative properties, PFAS are ubiquitous and are found in food and water . PFASs have come under recent scrutiny as bio-accumulating toxins; once they reach a certain concentration in the body, their hydrophobic long chain structures penetrate cellular lipid bilayers and displace and disrupt membrane structures. Concerningly,gallon pot in people aged 12 or older, PFAS chemicals including PFOS, PFOA, PFNA, and PFHxS have been detected in more than 95% participants of a ~7800 sample study in the U.S.. These chemicals have also been found to environmentally bio-accumulate with detection in several animals including mammals and aquatic species. The primary route of PFAS exposure in adults is through food and water ingestion.
There are also a few studies demonstrating toxicity of PFAS through skin exposure in human tissue engineered skin and in animals263–265. In US children , the exposure from dust ingestion and dietary ingestion is nearly equal258 . Bio-accumulation of PFAS results in multiple health detriments with exposure ranging from pre-natal time points into adulthood. Toxicity mechanisms of PFAS within the cell and on tissue development are poorly understood but implications have been made regarding disruption of viability and proliferative capacity, immune response, pro-estrogenic endocrine processes, lipid profile and fat development , cell membrane, endothelial barrier, gap junctions, and cytoskeleton . Higher PFAS concentrations in adults are associated with greater weight regain. Evidence for non-favorable lipid profiles linked to PFAS plasma concentrations has also been found including greater total cholesterol low-density-lipid , higher triglycerides, increased very low-density lipoprotein , and increased gamma glutamyl aminotransferase. Further, hormonal effects have been linked to PFAS compounds in adult subjects. Reproductive hormones such as sex-hormone binding globulin, follicle stimulating hormone, and testosterone concentrations have been found to be inversely related to PFOA and PFOS in people aged 12-30 years. Thyroid stimulating hormones and total T4 have been positively associated with PFAS while negative associations have been found with kidney function . Children have a higher burden of PFAS partially due to mouthing behaviors, their lower body size to area ratio, and possible exposure via breastfeeding . Studies with concerning outcomes concluded associations of increased PFAS exposure in utero and during childhood with adolescent/adult obesity, cholesterol, and increased beta cell dysfunction. Childhood obesity and overweight risk is in turn associated with higher adult risk of obesity and multiple chronic diseases including cardiovascular disease and diabetes. Several PFAS compounds are able to penetrate the placental barrier in pregnant women and reach fetal circulation. Although the mechanism of accumulation is not yet understood, multiple studies have shown that this transfer is preferential, that is, fetal concentration is higher than maternal concentration-possibly due to differences in fetal v. maternal blood.
Studies have associated higher maternal serum concentrations/exposure with decreased birthweight but increased infant adiposity. A growing number of studies have also linked high maternal serum concentrations of PFAS to increased pediatric/young adult adiposity and changes in lipid profiles. Hypothesized mechanisms of PFAS association with low birthweight and increased infant/childhood adiposity include lipid metabolism changes, reduced food/water intake by the mother, direct fetotoxic effect, endocrine disrupting effects, and other altered hormone levels. Much evidence points toward hormonal effects and their downstream interference of tissue development and function including body weight and adipose regulation. One direct example of hormone sensitive development effects include changes in anogenital distance which have been observed in female infants. Negative PFAS effects have also been explored with mechanisms acting on constitutive andostane receptor, pregnane-X receptor, estrogen receptor beta, and the phosphatidylinositol 3-kinase-serine/threonine protein kinase pathway. Hypothesized mechanisms for greater weight regain in adulthood include PFASs possible involvement in changing energy metabolism and homeostasis of thyroid hormones through transcriptional factor activation like peroxisome proliferator-activated receptors. PPARα and PPARγ are key regulators in fatty acid oxidation, differentiation, adipocyte proliferation and function, glucose breakdown, and lipid and lipoprotein metabolism. In mice, PFOA was shown to affect leptin and adiponectin release during differentiation of fat cells ; leptin is a regulator of energy homeostasis while adiponectin is secreted by mature adipocytes and effects insulin responsiveness. At this time, the high PFOA/PFOS exposure association with decreased birthweight is based on multiple conflicting literature. However, to shed light on conflicting conclusions, a case study that systematically reviewed 18 epidemiological and 21 animal studies regarding PFOA toxicity concluded that exposure to PFOA is in fact toxic to human reproduction and development300.
Conflicting evidence of the associations between PFAS maternal concentrations and childhood adiposity is also present, although a recent mass analysis of cohort studies has concluded an association with early life exposure to PFOA and increased risk for childhood obesity. Speculation upon the conflicting evidence of associations between PFAS maternal concentrations and childhood adiposity have been made regarding the differences in concentrations of PFAS evaluated in study populations and differences in method of subject weight measurement. In many studies PFAS associations with negative health effects were only observed in the highest fraction of serum concentration studied while in lower concentrations associations were not present. More evidence is required to fully understand PFAS associations and how they may vary with concentrations. It has been suggested that low maternal serum concentrations show positive associations with childhood adiposity and higher serum concentrations show positive, negative, or non-monotonic dose responses with childhood obesity; this evidence is supported by the fact that PFAS are endocrine disrupting chemicals 106, since EDCs have been shown to induce NMDR. Much of this association has been investigated due to concrete developmental effects PFASs have on rodent birthweight when exposed in utero. Importantly, animals have differences in PFAS metabolization and gestational duration when compared to humans and many animal effects were observed with very high levels of PFAS. Lipid profiles are associated with adult weight and prevalence of obesity. There is much conflicting evidence on whether PFAS plasma concentrations are associated with less favorable or beneficial outcomes on lipid profiles. Several studies have linked PFAS with higher total cholesterol, higher triglyceride levels,gallon nursery pot and higher low-density lipoprotein while other studies have linked higher PFAS plasma concentrations with beneficial lipid profile effects. Drawing conclusions based on adiposity effects of PFAS in animals should be carefully considered as well. Notably, mice have differences in adipocyte derived hormones; for example, resistin is secreted by adipocytes in the mouse but in humans is not expressed in adipocytes and is primarily present in monocytes and macrophages. Conflicting literature of PFAS and associations with health elucidate a need for further understanding at the cell and biological signaling level of PFAS effect on humans. These contradictions are exacerbated due to the poor understanding of the toxic mechanisms PFAS elicits. Several of these mechanisms share common modulators, the Hippo signaling pathway and the cell cytoskeleton. Hippo signaling is a crucial cellular pathway involved in organ development, growth, homeostasis, stem cell maintenance, and regeneration. Briefly, the classic Hippo cascade involves kinase regulators mammalian Ste20-like kinases 1/2 and large tumor suppressor 1/2 which act to regulate phosphorylation and degradation of Yes-associated protein and transcriptional co-activator with PDZ-binding motif. As downstream effectors of the Hippo pathway, YAP and its paralog TAZ target genes involved in cell growth, proliferation, differentiation and development. When Hippo is active, YAP/TAZ are phosphorylated leading to YAP/TAZ degradation and cytoplasmic retention. In the inactive state, YAP/TAZ accumulate in the nucleus and target proteins of TEA-domain containing family , runt-related transcription factor , and others .
Several factors can mediate activation of the Hippo pathway including the classic cascade involving Mst1/2 and Lats1/2, mechanical cues, cell polarity and adhesion mechanisms, metabolic pathways, ligand-dependent activation, and hormone/growth factor control. Further, YAP/TAZ are involved in pathway crosstalk as well. YAP and its core upstream regulators Mst1/2 and Lats1/2 have been investigated in several organs and their developmental dysregulation which may be linked to changes in body weight. Many organ systems have been studied to identify developmental problems after knockout of Mst1/2, Lats1/2, or YAP. Through these investigations, it has been shown that changes in the main regulators and effector YAP have caused lung epithelial defects, faults in kidney structure development, bone to fat ratio disruptions, over proliferation during intestinal development, improper regulation of liver development including epithelial and hepatocyte maturation changes , and pancreas mass/size changes. It is still unclear how PFAS may disrupt YAP regulation and how these mechanisms tie into body weight and organ development/size control. PFAS may regulate the Hippo pathway via mechanosensing extracellular matrix changes through focal adhesions and the actin cytoskeleton, a known Hippo pathway propagation. YAP/TAZ regulation through Rho/ROCK activation acts as a control mechanism for transcriptional control of cytoskeleton stability, PFAS is likely effecting cell function and viability through this Hippo pathway cascade as well. Adipogenic versus osteogenic polarization of mesenchymal stem cells is dependent on YAP/TAZ localization in the Hippo signaling pathway, a relationship linked through mechanical cues and cytoskeletal tension. An increase in nuclear YAP/TAZ localization corresponds to increased osteogenic stem cell differentiation while increases in cytosolic YAP/TAZ correspond to higher adipogenic differentiation. Mechanical regulation of adipogenesis upon nuclear localization has been suggested to work through transcription factor ß-catenin or SMAD proteins rather than TEAD. Due to the cytoskeletal involvement in adipose tissue, it is plausible that PFAS may dysregulate adipose through perturbation of cell cytoskeleton. PFAS chemicals have been found to act on the cell cytoskeleton through disruption of f-actin, microtubules, and gap junctions. PFAS has been shown to disrupt and fragment actin cytoskeleton and tight junctions in mice Sertoli cells and human microvascular endothelial cells. It is likely that PFAS effects cytoskeleton integrity and could change balance of osteogenic/adipogenic polarization of mesenchymal stem cells and/or adipose tissue homeostasis. During adipogenesis, cytoskeleton remodeling is a preliminary process and it has been found that the cytoskeletal components actin, tubulin, vimentin, and septin undergo localization and expression changes. Specifically, actin forms filament bundles in the cytoplasm of pre-adipocytes and short filaments in mature adipocytes with similar organization in the microtubules, and vimentin regulates lipid droplet accumulation by forming cage structures that surround lipid droplets. Septin has been found to form filaments or rings depending on timepoint within adipocyte differentiation. These findings support the cytoskeleton’s role in regulation of adipogenesis and lipid accumulation. In a study completed with rat cardiomyocytes, it was found that the adipokine, adiponectin acts on Rho/ROCK and increases RHO GTPase activity and induces cytoskeletal remodeling to further regulate glucose uptake and metabolism. Adiponectin effects weredemonstrated by its ability to increase membrane microvillar like protrustions, and increasing actin polymerization to form filamentous actin/actin stress fibers. Potentially PFAS chemicals may be directly disrupting the cytoskeleton or disrupting it indirectly through changes in the adipokine profiles that adipose tissue secretes. On the other hand, PFAS may regulate the Hippo pathway via mechanosensing extracellular matrix changes through focal adhesions and the actin cytoskeleton, a known Hippo pathway propagation. YAP/TAZ regulation through Rho/ROCK activation acts as a control mechanism for transcriptional control of cytoskeleton stability , PFAS could be effecting cell function and viability through this Hippo pathway cascade as well. The mechanical control of adipogenic differentiation of MSCs relies on both the integrity of the actin cytoskeleton itself and tension feedback from myosin II motor which directly acts on the Hippo signaling pathway.