The MET-FLAM Faculty

Grażyna KWAPISZEWSKA

Scientific Interests:

In addition, we have demonstrated the involvement of basement membrane components in the remodelling processes and recruitment of immune cells towards tissue injury [6, 7, 8]. Recently, we have focused on understanding how immune cells and their mediators influence cellular expansion and phenotypic switch of surrounding cells. Indeed, changes in immune cells composition actively effect vascular and parenchymal remodelling and thus disease progression. Immunomodulation can lead either to exacerbations of the disease or beneficial outcome [9, 10, 11, 12]. Thus, understanding the complex cellular intercommunication and metabolic alteration stay in the centre of scientific focus.

Proposed Dissertation Topic:

Background: Vascular abnormalities are a common feature of chronic lung diseases such as systemic sclerosis (SSc). These vascular abnormalities can manifest as remodeling and obliteration of pulmonary arteries, as seen in Ssc associated with pulmonary hypertension (PH). Current studies indicate that immune cells actively modulate behavior of underlying structural cells and are involved in disease progression. We have previously shown that transcription factor fos-related antigen-2 (Fra-2) is the master regulator of development of pulmonary vascular and parenchymal remodeling in an immunomodulatory manner [12, 13]. Moreover, increased immune cell numbers in Ssc Fra-2 transgenic (TG) mouse model positively correlated with vascular and parenchymal remodeling [12]. Parenchymal remodeling is due to expansion of fibroblasts and myofibroblasts and a decrease in lipofibroblast lineages, indicating that these cells respond differentially to the fibrotic milieu in the lung. Treatment of the animals with rosiglitazone or metformin restored lipid-laden fibroblasts and reverses experimental lung fibrosis [4, 5].

Hypothesis and objectives: Immune cells, by secreting a plethora of factors, can directly influence the metabolic status of underlying structural cells and thus lead to disease development. Therefore, we hypothesize that altered cross-talk between immune and structural cells leads to vasculopathy and to development of pulmonary fibrosis in Ssc Fra-2 TG mouse model. Using a wide array of molecular methodologies and animal models, we aim to investigate the influence of immune cells on the development of pulmonary vascular and parenchymal fibrosis.

Methods and approaches: In the 1st year the PhD candidate will perform isolation and characterization of different immune and structural cell populations from mouse and human lung tissue. He / she will conduct flow cytometry, cell-sorting and MACS approach, and multi-colour immunofluorescence staining to determine spatial localization of the immune cells and parenchymal cells in the healthy and fibrotic lung tissue. In the 2nd and 3rd year he / she will be dissecting the cross-talk between immune and structural cells (primary smooth muscle and fibroblast from healthy and diseased lung tissue from human and murine tissue) in co-culture systems and study the migration, proliferation and secretome of the structural cells. The receptor–ligand interaction will be analyzed in silico as well as in vitro. Spheroids and organoid models will be used to understand complex 3D interactions. Here, diverse immune cells will be injected in the spheroids / organoids and their effects on the expansion / distribution of the structural cells will be analyzed. Additionally, the metabolic status of the expanding cells will be established. In the 3rd and 4th year, the student will investigate which metabolic changes in the structural cells lead to the cellular expansion in vivo. Here, the lineage tracing mouse models [1, 2] will be applied to dissect expanding cells. Studies on human lung tissue will complement the variety of in-vitro and in-vivo approaches.

Pitfalls and alternative approaches: We will employ a range of methodologies to assess the metabolic status of the cells. For instance, we will utilize NMR spectroscopy (Nuclear Magnetic Resonance) to identify characteristic metabolic patterns and elucidate associated alterations in metabolic products. Additionally, mass spectrometry (MS) will be utilized for comprehensive metabolite profiling. The energy status of the cells will be evaluated using Seahorse XF technology. These methodologies are readily available at Med Uni Graz. By employing multiple analytical techniques, we will ensure robustness in our findings, mitigating potential limitations such as sensitivity issues encountered with low cell numbers. Furthermore, our organoid studies will benefit from collaboration with Prof. Susanne Herold from Giessen, Germany, who possesses expertise in this area. In circumstances where challenges arise with human organoids, particularly concerning iPSC differentiation, we will opt for mouse organoids due to their relative ease of cultivation.

Involved Faculty members: Grażyna Kwapiszewska (PI), Dagmar Kratky (metabolic phenotyping), Ákos Heinemann (immune cell isolation), Kathrin Eller (immune–structural cell cross-talk), Stefano Angiari (immunometabolism).

International Collaborations: Anne-Karine Perl (Cincinnati, USA), Edward Morrissey (Pennsylvania, USA), Norbert Weissmann (Giessen, DE), Elie-El Agha (Institute for Lung Health, Giessen, DE), Karin Tran-Lundmark (Lund, SE).

Facilities: Our team consists of five PhD candidates, four postdocs and two technicians. We are located on the newly opened basic research campus of the university, opposite the university hospital. Our lung group evolved from Ludwig Boltzmann Institute for Lung Vascular Research and is very closely working with the division of Pulmonology. We provide the required laboratory and office space, all necessary laboratory facilities, including cell culture, flow cytometry, multi-color immunofluorescence microscopy and animal laboratory equipment. Due to our close cooperation with German Lung Center we have also access to all cutting-edge technologies necessary for investigation of lung (patho)biology on the international level.

Preparatory Findings:

Figure 1: Fra2 overexpressing mice possess parenchymal and vascular remodelling which is associated with higher immune cell numbers.
Modulation of immune cells influence disease outcome and fibroblasts expansion. (Figure from [10]).

Figure 2: Modulation of fibroblast phenotype by FFA (free fatty acid) treatments shifts their properties towards less pathogenetic (less collagen and less PDGFRa production).
Unpublished observations.

1. Crnkovic S, Marsh LM, El Agha E, Voswinckel R, Ghanim B, Klepetko W, Stacher-Priehse E, Olschewski H, Bloch W, Bellusci S, Olschewski A, Kwapiszewska G: Resident cell lineages are preserved in pulmonary vascular remodeling. J Pathol, 2018 Apr;244(4):485-498.
   
2. Biasin V, Crnkovic S, Sahu-Osen A, Birnhuber A, El Agha E, Sinn K, Klepetko W, Olschewski A, Bellusci S, Marsh LM, Kwapiszewska G: PDGFRα and αSMA mark two distinct mesenchymal cell populations involved in parenchymal and vascular remodeling in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol, 2020; 318(4): L648–L697.
   
3. Crnkovic S, Valzano F, Fließer E, Gindlhuber J, Thekkekara Puthenparampil H, Basil M, Morley MP, Katzen J, Gschwandtner E, Klepetko W, Cantu E, Wolinski H, Olschewski H, Lindenmann J, Zhao YY, Morrisey EE, Marsh LM, Kwapiszewska G: Single-cell transcriptomics reveals skewed cellular communication and phenotypic shift in pulmonary artery remodeling. JCI Insight, 2022; 7(20):​e153471.
   
4. El Agha E, Moiseenko A, Kheirollahi V, De Langhe S, Crnkovic S, Kwapiszewska G, Szibor M, Kosanovic D, Schwind F, Schermuly RT, Henneke I, MacKenzie B, Quantius J, Herold S, Ntokou A, Ahlbrecht K, Braun T, Morty RE, Günther A, Seeger W, Bellusci S: Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis. Cell Stem Cell, 2017; 20(2):​261–273.e3.
   
5. Kheirollahi V, Wasnick RM, Biasin V, Vazquez-Armendariz AI, Chu X, Moiseenko A, Weiss A, Wilhelm J, Zhang JS, Kwapiszewska G, Herold S, Schermuly RT, Mari B, Li X, Seeger W, Günther A, Bellusci S, El Agha E: Metformin induces lipogenic differentiation in myofibroblasts to reverse lung fibrosis. Nat Commun, 2019; 10(1):​2987.
   
6. Veith C, Vartürk-Özcan I, Wujak M, Hadzic S, Wu CY, Knoepp F, Kraut S, Petrovic A, Gredic M, Pak O, Brosien M, Heimbrodt M, Wilhelm J, Weisel FC, Malkmus K, Schäfer K, Gall H, Tello K, Kosanovic D, Sydykov A, Sarybaev A, Günther A, Brandes RP, Seeger W, Grimminger F, Ghofrani HA, Schermuly RT, Kwapiszewska G, Sommer N, Weissmann N: SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension. Circulation, 2022; 145(12):​916–933.
   
7. Jandl K, Marsh LM, Hoffmann J, Mutgan AC, Baum O, Bloch W, Thekkekara-Puthenparampil H, Kolb D, Sinn K, Klepetko W, Heinemann A, Olschewski A, Olschewski H, Kwapiszewska G: Basement Membrane Remodelling Controls Endothelial Function in IPAH. Am J Respir Cell Mol Biol, 2020; 63(1):​104–117.
   
8. Hoffmann J, Wilhelm J, Marsh LM, Ghanim B, Klepetko W, Kovacs G, Olschewski H, Olschewski A, Kwapiszewska G: Distinct differences in gene expression patterns in pulmonary arteries of COPD and IPF patients. Am J Respir Crit Care Med, 2014; 190(1):​98–111.
   
9. Marsh LM, Jandl K, Grünig G, Foris V, Bashir M, Ghanim B, Klepetko W, Olschewski H, Olschewski A, Kwapiszewska G: The inflammatory cell landscape in the lungs of patients with idiopathic pulmonary arterial hypertension. Eur Respir J, 2018; 51(1):​1701214.
   
10. Birnhuber A, Crnkovic S, Biasin V, Marsh LM, Odler B, Sahu-Osen A, Stacher-Priehse E, Brcic L, Schneider F, Cikes N, Ghanim B, Klepetko W, Graninger W, Allanore Y, Eferl R, Olschewski A, Olschewski H, Kwapiszewska G: IL-1 receptor blockade skews inflammation towards Th2 in a mouse model of systemic sclerosis. Eur Respir J, 2019; 54(3):​1900154.
    
11. Birnhuber A, Jandl K, Biasin V, Fließer E, Valzano F, Marsh LM, Krolczik C, Olschewski A, Wilhelm J, Toller W, Heinemann A, Olschewski H, Wygrecka M, Kwapiszewska G: Pirfenidone exacerbates Th2-driven vasculopathy in a mouse model of systemic sclerosis-associated interstitial lung disease. Eur Respir J, 2022; 60(4):​2102347.
    
12. Jandl K, Marsh LM, Mutgan AC, Crnkovic S, Valzano F, Zabini D, Hoffmann J, Foris V, Gschwandtner E, Klepetko W, Prosch H, Flick H, Brcic L, Kern I, Heinemann A, Olschewski H, Kovacs G, Kwapiszewska G: Impairment of the NKT-STAT1-CXCL9 Axis Contributes to Vessel Fibrosis in Pulmonary Hypertension Caused by Lung Fibrosis. Am J Respir Crit Care Med, 2022; 206(8):​981–998.
    
13. Birnhuber A, Biasin V, Schnoegl D, Marsh LM, Kwapiszewska G: Transcription factor Fra-2 and its emerging role in matrix deposition, proliferation and inflammation in chronic lung diseases. Cell Signal, 2019; 64:​109408.