This study sought to further determine the crosstalk between TERT and autophagy on microvascular function using a genetic rodent mutant model. There are a number of salient findings from these experiments. Primarily, genetic mutation of TERT results in a switch in the mechanism of microvascular vasodilation from NOS to H2O2, similar to that observed in arterioles from patients with CAD. This switch in vasodilator mechanism is paralleled by an increase in mitoPY1 fluorescent signal, a reduction in shear-induced NO production, and a reduction in shear-induced autophagic flux relative to WT rats. Importantly, activation of autophagy with the natural disaccharide, trehalose, for 28 days reversed the switch in dilator mechanism, abolished differences in both shear-induced H2O2 and NO production, in addition to differences in shear-induced autophagy. Taken together, these findings highlight the crucial independent and synergistic roles of autophagy and TERT in maintaining healthy microvascular endothelial function.

Our lab has previously established the important role that TERT holds in maintaining microvascular function in both animal and isolated human vessels. We have previously established that genetic loss of the catalytic subunit of telomerase (TERT), but not the RNA component (TERC) is associated with a pathological switch in the mechanism of vasodilation in a murine model [2]. Pharmacological inhibition of TERT in otherwise healthy isolated human adipose and coronary resistance arterioles [5] results in a pathological switch in the mechanism of microvascular vasodilation to shear stress from NO to H2O2, similar to what is observed in adipose resistance and atrial arterioles from patients with CAD. On the other hand, pharmacological activation of TERT rescues NO-mediated vasodilation in patients with CAD [5]. Additionally, genetic inactivation of TERT in a murine model is associated with a greater formation of ROS, exacerbation of microvascular dysfunction, and larger infarct sizes in response to angiotensin-II relative to WT or matched littermates that lack the RNA component of TERT [1, 2], while the location of TERT is important in mediating this response as mitochondrial translocation of TERT suppresses ROS formation and loss of mitochondrial TERT results in a significant reduction of ATP production [1, 4, 5]. In this context, TERT is positioned as a crucial regulator of microvascular and mitochondrial health.

Previously, we demonstrated that microvascular function in isolated human resistance arterioles is in part regulated by a crosstalk between autophagy and telomerase [13]. This was demonstrated by manipulating both autophagy and telomerase independently and in conjunction with one another. In the context of CAD, both autophagy and telomerase are reduced, resulting in a microvascular phenotype wherein H2O2 predominates as the primary mechanism of vasodilation to shear stress. This was corroborated with a reduction in shear-induced autophagic flux [13]. Within the current study, the pathological phenotype typically observed with CAD was mimicked using a rodent model of TERT inactivation. This was paralleled by an overall reduction in TERT mutant rats of the fluorescent signal of Lysotracker Red DND-99, used to track autophagy in response to shear stress. This is taken as evidence of a reduction in autophagic flux within TERT mutant rats. Furthermore, in agreement with our previous work, we observed an increase in mitochondrial H2O2 production in response to shear stress, and a significant reduction in shear-induced NO production in TERT mutant rats relative to WT. These findings were corroborated by a reduced protein expression of eNOS within mesenteric arteries from TERT Mutant rats. This is taken as further evidence that TERT mutation/inactivation impairs autophagic flux, and in turn, NOS signaling within the microvasculature, impacting functional responses to shear stress. Interestingly, when separated by sex, there were no differences in shear-induced mitochondrial H2O2 or NO production.

Similar to our previous work in isolated lungs of WT and TERT mutant rats [6], there were no differences in markers of autophagy or mitophagy (LC3B, p62, Beclin 1, Pink 1, Parkin) within isolated aortas. However, within the current study we specifically measured autophagic flux, or the rate of autophagy via the addition of BAFA1 to aortas (protein expression). This is in contrast to the Lysotracker Red DND-99 findings within the mesenteric arteries and can be taken as evidence that while there are no differences between groups in static conditions, TERT mutation impacts the resiliency or responsiveness of the vasculature to respond to dynamic conditions (e.g. shear stress).

Given that inactivation of TERT resulted in a switch from NO to H2O2, and impaired shear-induced autophagic flux, we hypothesized that activation of autophagy would rescue this phenotype. Previous evidence in both mice and humans has demonstrated that activation of autophagy with the natural disaccharide trehalose ameliorates age-associated vascular dysfunction [15, 16]. It is important to note that trehalose does have caloric content (~ 16 kJ/g). In the context of previous human studies examining vascular function, the beneficial vascular effects were dependent upon the amount of weight gained across the supplementation period, as subjects ingested 100 g of trehalose per day, while maintaining their regular diet [15]. This resulted in an excess of approximately 400 kcal per day over the course of 12 weeks, and the beneficial effects on vascular function were isolated to those subjects who gained no more than 2.3 kg over the course of 12 weeks. The amount given within the current study (2% w/v, 10 g in 500 mL) was well below what previous human studies have administered but is in line with previous animal studies [8, 16, 21, 24]. The novel finding of the current study is that activation of autophagy with trehalose rescued NO-mediated microvascular FMD despite a genetic inactivation of TERT. Furthermore, activation of autophagy with trehalose did not impart negative effects in WT rats, as NO-mediated vasodilation was maintained. Interestingly, and in contrast to our hypothesis, activation of autophagy did not alter mitochondrial H2O2 production or NO production in response to shear stress. Taken together, autophagy regulates vasomotor responses to shear stress and can improve pathological phenotypes as a result of TERT deficiency.

There are several experimental considerations that warrant discussion. First, as mentioned previously, the genetic inactivation of TERT was not a total abolition. Instead, the protein is still apparent while telomerase activity as measured by the TRAP assay is significantly reduced below background. Second, it was not within the scope of the study to determine the mechanism by which trehalose activates autophagy, however, strong evidence from other laboratories has indicated that trehalose supplementation results in nuclear translocation of Transcription Factor EB, which is the master regulator of transcription of autophagy-related genes and lysosomal function [9, 10, 20, 23]. Third, the present experiments focused on isolated resistance arteries, they were performed in a systemic TERT loss-of-function model combined with chronic in vivo autophagy modulation. The switch from NO to H₂O₂ as the mediator of FMD recapitulates what is observed in patients with coronary artery disease, underscoring translational relevance [13]. Moreover, previous studies have established in vivo consequences of TERT deficiency or enhancement in cardiovascular disease. For example, telomerase deficiency predisposes to larger infarcts and impaired recovery after ischemia–reperfusion injury [1, 3], while mitochondrial TERT confers cardioprotection by improving complex I activity and reducing infarct size [4]. Take together, these reinforce the physiological importance of TERT in cardiovascular pathology. Nonetheless, we acknowledge that our study did not directly assess outcomes in atherosclerosis or myocardial infarction models, and future work is needed to determine whether autophagy activation can translate into protection in these disease contexts. Finally, while both male and female rats were included, the study was not designed or powered to detect sex-based differences in microvascular responses. The observed effect sizes for mitochondrial H₂O₂ (MitoPY1) and nitric oxide (NO) fluorescence were 0.45 and 0.73, respectively. Achieving 80% statistical power at α = 0.05 would require approximately 158 and 62 animals for these outcomes, which was beyond the ethical and logistical scope of the present investigation. Accordingly, analyses stratified by sex are presented as exploratory and should be interpreted with caution. Importantly, the primary outcomes regarding the mechanism of flow-mediated dilation and the effects of autophagy activation were adequately powered to test the central hypothesis of the study.