Human Aortic Fibroblasts Research Articles

Cyborg Vessel

Cyborg Vessel

Electronic Blood Vessel

Summary Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface.

Abstract 9648: Aldosterone Increases Transforming Growth Factor-β Expression to Activate Autophagy and Increase Aortic Fibroblast Collagen Synthesis and Vascular Stiffness

Elevated levels of aldosterone (ALDO) are associated with aortic remodeling and increased arterial stiffness; however, the role of fibroblasts in these processes remains unknown. Accordingly, human aortic fibroblasts (HAoF) were treated with ALDO (10 -9 -10 -7 mol/L) or a vehicle control (V) for up to 72 h. Compared to V, ALDO increased HAoF expression of transforming growth factor-β (TGFβ) in a dose- and time-dependent manner with a maximal increase of 2.9 ± 0.3-fold (p<0.01) at 72 h. This change was associated with a 252.6 ± 28.8 % (p<0.01) increase in collagen III protein levels in ALDO-treated HAoF; these findings were confirmed by immunohistochemistry. Increased TGFβ expression was also associated with an increase in reactive oxygen species generation (126.4 ± 15.6 vs. 435.5 ± 38.6 fluorescent units, p<0.01) and activation of autophagy as demonstrated by increased beclin1 expression (211.3 ± 23.2% vs. V, p<0.01), lipidation of LC3BI to LC3BII (p<0.01) and autophagic flux assay. To demonstrate that autophagy was necessary to increase collagen III expression, HAoF were transfected with beclin1 siRNA to decrease expression by 69%. Inhibition of autophagy led to a 78% decrease (p<0.01) in collagen III expression without influencing TGFβ levels. To confirm these findings in vivo , C57Bl/6 mice received ALDO (50 μg/kg/d) or V for 21 days. In ALDO-treated mice, aortic TGFβ and LC3BII expression were increased with a concomitant 3.5 ± 0.5-fold (p<0.01) increase in collagen deposition. Pulse wave velocity (PWV), a marker of arterial stiffness, was increased in ALDO-treated mice (288.4 ± 22.4 vs. 389.6 ± 19.8 cm/sec, p<0.01). Treatment with imatinib (40 mg/kg/d) for 14 days downregulated vascular TGFβ expression, diminished collagen deposition, and decreased PWV by 41.1 ± 9.8% (p<0.01). To determine the clinical relevance of these findings, human aorta segments from patients with atherosclerotic vascular disease had higher levels of TGFβ, LC3BII, and fibrosis compared to segments from subjects without disease. These data indicate that ALDO activates fibroblasts to modulate vascular collagen deposition and increase vascular stiffness through a mechanism that involves, in part, TGFβ-mediated activation of autophagy in fibroblasts.

Aldosterone Activates Autophagy To Increase Fibroblast Collagen Synthesis and Vascular Stiffness

Hyperaldosteronism is associated with increased arterial stiffness; however, the contribution of fibroblasts to this response remains unknown. Accordingly, we treated human aortic fibroblasts (HAoF) with aldosterone (ALDO) (10−7 mol/L) for up to 72 h or vehicle control (V). Compared to V, ALDO increased transforming growth factor‐β (TGFβ) expression by 2.5 ± 0.2‐fold (p<0.01) and collagen III levels by 252% (p<0.01) in a time‐dependent manner. Increased TGFβ expression was associated with activation of autophagy as shown by lipation of LC3BI to LC3BII (p<0.01). To demonstrate that autophagy modulated collagen expression, autophagy was inhibited by transfecting cells with beclin1 siRNA. Inhibition of autophagy led to a 78% decrease (p<0.01) in collagen III expression without influencing TGFβ levels. To confirm these findings in vivo, C57Bl/6 mice were infused with ALDO (50 μg/kg/d) or V for 21 days. In ALDO‐treated mice, aorta TGFβ and LC3BII expression were increased with a concomitant 3.5 ± 0.5 ‐ fold (p<0.01) increase in perivascular collagen deposition. This was associated with an increase in pulse wave velocity (288.4 ± 22.4 vs. 389.6 ± 19.8 cm/sec, p<0.01), a marker of arterial stiffness, in ALDO‐treated mice. These data indicate that ALDO increases perivascular collagen deposition and vascular stiffness through a mechanism that involves activation of autophagy in fibroblasts.

Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple‐transcript transgene expression in mammalian cells and microtissues

Adenoviral particles can efficiently transduce a broad spectrum of cell types, so they are widely used in basic research and clinical trials. We have developed a novel adenoviral vector platform for delivery of constitutive or streptogramin-inducible expression of up to three therapeutic transgenes into a variety of murine and human cell lines, primary cells and microtissues. Coordinated expression of three independent transgenes in a compact genetic format was achieved by two different expression configurations: (i) The multicistronic expression format consisting of a single constitutive (simian virus 40 promoter, P(SV40); murine or human cytomegalovirus immediate-early promoter, P(mCMV), P(hCMV)) or regulated (streptogramin-inducible) promoters (P(PIR)ON2) driving the expression of a single multicistronic transcript of which the first cistron is translated in a cap-dependent manner and the two subsequent ones by internal ribosome entry site (IRES)-mediated translation initiation. (ii) The triple-transcript expression configuration, in which a combination of well-established (P(SV40), P(hCMV), P(mCMV)) and novel synthetic constitutive promoters (P(GTX)) control transcription of three expression units. The constitutive multigene expression design enabled coordinated high-level expression of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY), the human vascular endothelial growth factor 121 (VEGF(121)) and the human placental secreted alkaline phosphatase (SEAP) in monolayer populations and microtissues of Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary neonatal rat cardiomyocytes (NRCs) and primary human aortic fibroblasts (HAFs). Streptogramin-inducible tricistronic SAMY-VEGF(121)-SEAP expression provided excellent regulation performance-high-level induction in the presence of the streptogramin antibiotic pristinamycin I (PI), near-undetectable basal expression in the absence of PI, optimal adjustability and perfect reversibility-in all cell types, in particular in NRCs and NRC-derived myocardial microtissues. Triple-transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size-constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios.

A novel binary adenovirus‐based dual‐regulated expression system for independent transcription control of two different transgenes

Stringent multitransgene control is a prerequisite for future gene-therapy and tissue-engineering scenarios and requires constant improvements in design to achieve optimal conditional transcription profiles. We have pioneered a variety of recombinant adenoviruses which (i) enable streptogramin-responsive transgene transduction in a compact autoregulated one-virus format, (ii) manage independent streptogramin- and tetracycline-responsive control of two different transgenes from a single divergent expression unit, and (iii) control sense and antisense expression of the human cyclin-dependent kinase inhibitor p27(Kip1) to engineer conditional positive (enforced S-phase entry, p27(Kip1)-antisense expression) or negative (G1-phase-specific growth arrest, p27(Kip1)-sense expression) growth control in mammalian cell lines and human primary cells. The transgene control performance of all adenoviral expression configurations has been rigorously optimized for tight, balanced and maximum expression levels and was validated for intracellular as well as for secreted product in a variety of biotechnologically relevant cell lines (Chinese hamster ovary cells [CHO-K1], baby hamster kidney cells [BHK-21]) as well as in human cell lines (human fibrosarcoma cells [HT-1080]) and primary cells (human aortic fibroblasts [HAFs]). We believe that multiregulated multigene-controlled adenoviruses are important assets for successful therapeutic reprogramming of mammalian cells in clinically relevant scenarios.