Human arteries engineered in vitro

Abstract

There is a pressing need to develop methods to engineer small-calibre arteries for bypass surgery. We hypothesized that the rate-limiting step that has thwarted previous attempts to engineer such vessels from non-neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non-neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

Figure 1

Telomerase activity in smooth-muscle cells increases lifespan and extends telomeres. (A) Stable infection of smooth-muscle cells (SMCs) with a retroviral vector encoding the human telomerase reverse transcriptase subunit (hTERT) yielded hTERT-SMCs (hT) that were positive for telomerase activity, whereas control uninfected SMCs (un) and vector-infected SMCs (v) were negative. Human embryonic kidney cells that expressed hTERT (Armbruster et al., 2001) were used as a positive control (Ctl). (B) Southern hybridization shows telomere erosion in control uninfected (un) and vector-infected (v) SMCs, whereas hTERTsMCs extend telomeres for at least 80 population doublings (PDs). (C) Control uninfected (un) and vector-infected (v) SMCs senesced at PD 37, whereas hTERT-SMCs grew until at least PD 100. The asterisk indicates the PCR internal control. HI, heat inactivated.

Figure 2

hTERT-SMCs are phenotypically normal. Smooth muscle cells (SMCs) were stably infected with a retroviral vector encoding the hTERT human telomerase reverse transriptase subunit (hT). As controls, SMCs were also stably infected with a control retroviral vector (v) or were left uninfected (un). Both late-passage hTERT-infected SMCs and the control SMCs (A) maintained protein expression of the differentiation markers calponin and smooth-muscle myosin heavy chain (SM-MHC) and the extracellular matrix protein tropoelastin, (B) downregulate hyperphosphorylated retinoblastoma protein (pRb) in high-density cultures, (C) upregulate p53 protein in response to DNA damage by ultraviolet irradiation, (D) did not overexpress c-MYC protein, (E) lacked endogenous hTERT messenger RNA and (F) expressed p16 protein. (G) SMCs stably infected with a retroviral vector encoding hTERT also had a normal karyotype. For immunoblot or RT–PCR (PCR after reverse transcription) analysis, actin or glyceraldehyde-3-phosphate dehydrogenase (GADPH) served as a loading control, respectively. 293T cells served as a positive control and water as a negative control in RT–PCR assays for endogenous hTERT expression. C, confluent cultures; ectop. hTERT, ectopic hTERT; endog. hTERT, endogenous hTERT; PDs, population doublings; SC, subconfluent cultures; UV+ or −, treatment with or without ultraviolet irradiation.

Figure 3

Telomerase enables robust vascular tissue engineering. Haematoxylin–eosin staining of smooth-muscle cells (SMCs) uninfected (A) or stably infected with a retrovirus encoding the hTERT human telomerase reverse transcriptase subunit (B). TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) staining of vessels derived from (C) uninfected SMCs shows widespread cell death seen as dark-staining nuclei in contrast to (D) vessels engineered with SMCs expressing hTERT. (E) Immunostaining for von Willebrand Factor shows a confluent monolayer of endothelial cells lining the lumen (^*) of an hTERT-SMC vessel. (F) Immunoblots showing that all four hTERT-SMC vessels engineered maintain expression of protein characteristics of differentiated SMCs, and this expression is similar to that seen in vessels derived from uninfected SMCs. Scale bars, 0.5 mm (A,B); 100 µm (C–E). Actin was used as a loading control. SM-MHC, smooth-muscle myosin heavy chain.