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Hyperproliferative smooth muscle (SMC)-like cells harboring mTOR mutations invade lungs in Lymphangioleiomyomatosis (LAM) leading to respiratory failure. We posit that, unlike healthy SMCs, which stabilize fragile neovessels, LAM-SMCs promote disruption of microvessels by inducing endothelial cell (EC) apoptosis. HiPSCs from LAM or healthy subjects were differentiated into iSMCs and co-cultured with ECs on Matrigel. Healthy iSMCs enhanced EC network persistence (?72H). However, LAM-iSMC co-culture led to rapid network collapse (<15H), associated with increased expression of vessel-destabilizing factor, ANGPT2, and decreased expression of eNOS, TIE2, CD31 (5-8 fold, p<0.05) over 24-72H. Notably, the beneficial effects of healthy iSMC co-culture on EC network formation and stability were abrogated by addition of LAM-iSMC conditioned media (CM), assessed by live cell imaging. Similarly, LAM-iSMC CM increased EC apoptosis (2-3 fold, p<0.05) at 24-72H, assessed by Annexin V/PI flow cytometry and cleaved Caspase-3, -9 protein expression. Notably, Mitotracker studies showed that mitochondria (Mt) were abundantly shed into the CM by iSMCs. While Mt isolated from healthy iSMCs had no detrimental effect on EC survival and proliferative ability, LAM-iSMCs? Mt led to increased ANGPT2expression, EC apoptosis (2-fold, p<0.05) and reduced EC gene and protein expression. Apoptotic ECs are known to release exosomes (EXOs) containing translationally controlled tumor protein (TCTP), an upstream positive regulator of the mTOR cascade. Intriguingly, LAM-iSMCs internalized EXOs derived from apoptotic EC, which corroborated with increased levels of proliferation-associated phospho-mTOR-and-S6k protein. Moreover, inhibition of EXO release from apoptotic ECs by pre-treatment with GW4869 decreased phospho-mTOR-and-S6k activity in LAM-iSMCs, consistent with a role for EXO-derived TCTP in amplifying the hyperproliferative phenotype of LAM-iSMC. Thus, cytotoxic Mt released from LAM-iSMCs mediate EC network destabilization and apoptosis. Conversely, uptake of TCTP-containing EXOs from ECs fuels LAM-SMC growth, providing important insights into a novel ?feed-forward? mechanism possibly underlying lung vascular destruction in LAM disease.

Bioartificial lungs created using autologous induced pluripotent stem cell (iPSC) products may circumvent graft rejection to treat severe respiratory diseases. In vitromaturation of iPSC-derived lung epithelial cells is critical to recreate an efficient air-blood barrier (ABB), requisite for mediating lung function. We posit that crosstalk between lung progenitors (LPs) and endothelial cells (iECs) can recapitulate establishment of an ABB. Human iPSCs were differentiated into CD144+VEGFR2+iECs and NKX2.1+EpCAM+LPs, confirmed by flow cytometry and immunocytochemistry. In 2-D cultures, LPs exposed to small extracellular vesicles (sEVs) purified from iECs increased distal/alveolar (AQP5, PDPN, SFTPC) epithelium expression (all ?2-fold; p<0.05). Inhibition of iEC-sEV production diminished this response by ?50%. In addition, iECs were also directly co-cultured with LPs for 24-72hrs, prior to CD144 immunomagnetic separation; in parallel, iEC monoculture acted as a control. EC-associated gene and protein changes were quantified by Q-PCR and Western blots, respectively. At 24hrs of co-culture, the angiopoietin-1 (ANG1) and ANG2 ratio (ANG1:ANG2) in CD144+iECs was significantly decreased by <53?6% (p<0.05), implying vessel destabilization. This correlated with reduced Mst1 kinase expression and YAP1 activation - a key mediator of Hippo signaling involved in angiogenesis. At 48hrs, the expression of mature EC markers, VEGFR2 and CD105, were increased by ?2-fold compared to control. At 72hrs, the ANG1:ANG2 was reversed (?3-fold increase), with augmented expressions of PECAM and ICAM (?2-fold), vascular tight junction proteins involved in ABB permeability, compared to earlier time-points or control (all p<0.05). In conclusion, our results provide novel insight into how iECs guide distal airway maturation by sEV-mediated mechanism. Reciprocally, LPs regulate iEC behavior initially by inducing vessel plasticity and angiogenesis; subsequently, enhancing EC maturation and endothelial barrier integrity. Thus, cell-based strategies that harness the dynamic interplay between LPs and ECs could promote better alveolar re-endothelialization and improve the robustness of ABB in bioengineered lung matrices for regenerative therapies.

Introduction:Lymphangioleiomyomatosis (LAM) is a lethal neoplasm resulting from inactivation of TSC1/2genes, leading to aberrant mTOR signalling. Lung invasion and airway destruction by hyper-proliferative smooth muscle-like (SMC) LAM cells results in pulmonary failure. We hypothesize that LAM cells exert a deleterious effect on lung progenitors (LPs) that is dependent on mTOR activity, and mediated by the release of toxic small extracellular vesicles (sEVs).Methods/Results:Induced pluripotent stem cells (iPSC) were derived from healthy subjects or LAM patients and differentiated into ?-SMA+CNN1+iSMCs and CD47hiEpCAM+LPs using stage-specific morphogens (85?5% and 82?7% purity, respectively). Compared to sEVs purified from healthy iSMC conditioned-medium, sEVs from LAM-iSMCs decreased LP survival (44?7% vs. 87?8%), increased apoptosis (55?8% vs. 11?4%) assessed by annexin V/PI staining, and reduced cell cycle progression (23?5% vs. 55?4% in S-phase) as determined by EdU/Sytox incorporation (all p<0.05). Since translationally controlled tumor protein (Tpt1) positively regulates mTOR, LAM-iSMCs were transduced with Tpt1shRNA to investigate its indirect role in LP survival. Knockdown (KD) of Tpt1(?55% KD of gene and protein expression) significantly repressed mTOR activity in LAM cells, as evidenced by 48?6% decrease in phospho-mTOR protein levels. Strikingly, the detrimental paracrine effects of LAM-sEVs on LPs were reversed following Tpt1KD, with improved viability (67?8%), reduced apoptosis (31?5%), and augmented cell replication (45?6%; all p<0.05). Three-dimensional organoid assays (75 days) demonstrated that LPs exposed to healthy iSMC-sEVs yielded bronchiolar organoids comprised of complex folding patterns. In contrast, LPs cultured with LAM-sEVs produced smaller organoids that exhibited simplified cystic bronchiolar morphology, mimicking the typical human LAM lung pathology. Of note, this effect of LAM-iSMCs sEVs on LP development was abolished post-Tpt1KD.Conclusion:Endogenous Tpt1expression contributes to the destructive effect of LAM-iSMC secretome on lung airway progenitors, in large part attributable to mTOR-dependent release of sEVs. Thus, Tpt1may be a novel therapeutic target for LAM disease.

Introduction:Typically, induced pluripotent stem cells (iPSC) undergo lineage differentiation on commercially available matrices (e.g. Matrigel). Since the niche dynamically regulates stem cell behaviour, we exploited human lung extracellular matrices (ECM) derived from healthy ?never smoker? subjects (NS) or patients with chronic obstructive pulmonary disease (COPD) as potentially more authentic platforms to model the growth of vascular and lung progenitors (LP).Methods/Results:Lungs harvested from age-matched NS or COPD patients were decellularized and lyophilized to prepare lung ECM. Human iPSCs were differentiated into CD144+VEGFR2+endothelial cells (iECs), ?-SMA+CNN1+smooth muscle cells (iSMCs), and NKX2.1+Sox2+Sox9+LPs, confirmed by immunocytochemistry and flow cytometry. Live cell imaging showed that iECs and iSMCs cultured on Matrigel or NS ECM aligned closely to form stable vessel networks. However, when grown on COPD ECM, they exhibited clustering and poor vessel assembly. This correlated with >2-fold increase in ANG2 levels, suggesting endothelial activation and vessel destabilization. Phalloidin staining revealed that LPs cultured on Matrigel or NS ECM displayed organized epitheloid morphology, whereas those grown on COPD ECM appeared elongated, reflecting stress-induced actin cytoskeletal changes. In addition, COPD ECM decreased LP viability (Annexin V/PI) and replication (EdU/Sytox) by 53?8% and 32?9% (p<0.05), respectively, relative to NS. Mainly proximal lung epithelial differentiation of LPs was seen on Matrigel and lung ECMs; only NS ECM increased the alveolar markers, SCBG1A1and SFTPC(?2-fold) vs. Matrigel, indicating greater distal airway patterning. Notably, LPs grown on COPD ECM exhibited elevated expression of secretory markers, MUC5AC, P63, and SFTPD(?2-fold), with ~40% reduction in NKX2.1and FOXJ1(all p<0.05) compared to NS/Matrigel, mimicking the secretory phenotype of COPD lungs.Conclusion:Human lung ECMs effectively model healthy and pathological niches. Thus, disease-specific lung matrices may be harnessed to optimize cell-based strategies for lung repair/regeneration and as drug screening platforms to assess the effect of small molecules on vascular and LP differentiation.

In Lymphangioleiomyomatosis (LAM), mTOR mutations result in hyperproliferative smooth muscle-like cells (LAM-SMCs) that invade and destroy lung alveolar and vasculature structure. mTOR inhibitors, like rapamycin, are cytostatic and not curative, highlighting the need for novel therapeutic targets. Translationally controlled tumor protein (Tpt1) is an upstream positive regulator of mTOR signalling that is highly expressed by LAM-SMCs relative to healthy SMCs. Thus, we posit that Tpt1inhibition will decrease LAM-SMCs proliferation. LAM-iSMCs were derived from LAM iPSCs. Knockdown (KD) of Tpt1expression was achieved by transduction with a selective siRNA, compared to a scrambled control (SC). Tpt1-KD was confirmed in LAM-iSMC by Q-PCR and WB (?50%). Compared to SC, Tpt1-KD resulted in cell cycle arrest at G0/G1 (41.5% vs. 67. 4% respectively, p<0.05) and a reduction in LAM-iSMC proliferation (49.6% vs. 23% respectively, p<0.05), assessed by Edu incorporation (flow cytometry). Given that we have previously shown that LAM-iSMCs disrupt endothelial cells (EC) networks by secretion of cytotoxic exosomes and mitochondria (Mt), we investigated if Tpt1-KD reduced the destructive effect of LAM-iSMC?s secretome. Compared to conditioned media (CM) from SC controls, ECs cultured with CM from Tpt1-KD LAM-iSMCs demonstrated increased EC viability (33% vs. 64.2% respectively, p<0.05) and reduction in apoptosis (49% vs. 23% respectively, p<0.05) as assessed by Annexin V/PI flow cytometry. Notably, EC showed markedly reduced uptake of LAM-iSMC Mt from Tpt1-KD-CM compared to SC-CM, associated with reduced EC apoptosis (18.93% vs. 25.3%, KD vs. SC respectively, P<0.05). Moreover, Tpt1-KD blunted the inhibitory effect LAM-iSMCs-derived exosomes on EC-associated gene and protein expression compared to SC controls. Importantly, GW4869-mediated inhibition of exosome release from Tpt1-KD LAM-iSMCs produced an even greater improvement in EC associated protein expression, cell viability and survival. In conclusion, endogenous Tpt1expression contributes to the hyperproliferative and neoplastic phenotype of LAM-SMCs and potentiates the release of EC-toxic exosomes and Mt. Thus, Tpt1inhibition may provide a novel therapeutic target for LAM treatment.