Anna A. Birukova, MD

1. Crosstalk between microtubules and microfilaments in endothelial barrier regulation.

Much of my research career has been directed at understanding molecular regulation of lung endothelial permeability and inflammation with a focus on the role of microtubules (MT) and microtubule-associated guanine nucleotide exchange factors in the regulation of small GTPases, microtubule dynamics and lung endothelial cell (EC) barrier function. In early studies I discovered that pharmacologic disassembly of MT in human pulmonary EC caused barrier dysfunction triggered by small GTPase Rho, which resulted in the remodeling of actin cytoskeleton, initiation of cell contraction, and increased endothelial permeability. I then hypothesized that MT dynamics may be also involved in EC permeability induced by circulating vasoactive agonists. We were the first to demonstrate the disassembly of peripheral MT by thrombin, which stimulated Rho signaling and EC barrier disruption. Looking into mechanisms of Rho activation by MT disassembly, we found that this mechanism is driven by MT-bound Rho-specific guanine nucleotide exchange factor, GEF-H1. These pioneering studies showed that partial disassembly of peripheral MT pool causes cytoplasmic release and activation of GEF-H1, therefore causing activation of barrier-disruptive Rho pathway. In turn, MT structure can be stabilized by elevation of cyclic-AMP levels, which leads to suppression of Rho signaling and EC permeability. Further investigation demonstrated that phosphorylation of MT regulator protein stathmin is a key mechanism of MT stabilization by cAMP. These and further studies by my group have shown that MT-dependent activation of Rho pathway is a fundamental mechanism of endothelial barrier permeability triggered by various vasoactive and pro-inflammatory (such as TGFβ and TNFα) agonists which appear during acute lung injury.

a. Birukova AA, Smurova K, Adyshev D, Kaibuchi K, Alieva I, Garcia JG, Verin AD. Novel Role of microtubules in thrombin-induced endothelial barrier dysfunction. FASEB J, 2004 18(15):1879-1890

b. Birukova AA, Smurova K, Birukov KG, Usatiuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva I, Garcia JG, Verin AD. Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: Role of Rho-dependent mechanisms. J Cell Physiol, 2004 201(1):55-70.

c. Poroyko V, Birukova AA. ARHGEF2 (rho/rac guanine nucleotide exchange factor (GEF) 2). Atlas Genet Cytogenet Oncol Haematol. Elsevier, 2007.

d. Tian X, Tian Y, Sarich N, Wu T, Birukova AA. Novel role of stathmin in microtubule-dependent control of endothelial permeability. FASEB J, 2012 Sep;26(9):3862-74.

2. Microtubule-associated signaling involved in the control of lung inflammation and injury.

Since inflammation is an integral part of ALI and has a major impact on lung function, I focused on in vitro and in vivo models of lung injury induced by Gram-positive and Gram-negative bacterial compounds. We examined hierarchy of stress kinase pathways and Rho signaling in inflammatory and permeability response by lung micro- and macrovascular EC. We also demonstrated the mechanism of redox-sensitive Rho activation by LPS via GEF-H1 activation leading to pulmonary EC dysfunction and lung inflammation. I was also excited to add a new dimension to my research program and explore a role of mechanical microenvironment in control lung vascular inflammation. We described a novel mechanism of stiffness-dependent exacerbation of vascular inflammation and escalation of ALI via stimulation of GEF-H1 expression. In turn, inhibition of local stiffening of lung vasculature attenuated the lung injury. This pathway represents the previously unknown mechanism of positive feedback regulation of inflammation.

a. Wu T, Xing J, Birukova AA. Cell-type-specific crosstalk between p38 MAPK and Rho signaling in lung micro- and macrovascular barrier dysfunction induced by Staphylococcus aureus-derived pathogens. Transl Res. 2013 Jul;162(1):45-55.

b. Kratzer E, Tian Y, Sarich N, Wu T, Meliton A, Leff A, Birukova AA. ROS-mediated microtubule destabilization triggers GEF-H1-dependent endothelial permeability and inflammation in septic lung injury. Am J Resp Cell Mol Biol, 2012 Nov;47(5):688-97.

c. Mambetsariev I, Tian Y, Wu T, Lavoie T, Solway J, Birukov KG, Birukova AA. Stiffness-activated GEF-H1 expression exacerbates LPS-Induced lung inflammation. PLoS One. 2014 Apr 16;9(4):e92670.

d. Meng F, Mambetsariev I, Tian Y, Beckham Y, Meliton A, Leff A, Gardel ML, Allen MJ, Birukov KG,  Birukova AA. Attenuation of LPS-induced lung vascular stiffening by lipoxin reduces lung inflammation. Am J Resp Cell Mol Biol, 2015 Feb;52(2):152-61.

3. Search for protective agonists for ALI/ARDS treatment.

In search for protective agonists for ALI treatment, I investigated natural compensatory mechanisms critical for the lung barrier protection under life-threatening conditions and studied effects of atrial natriuretic peptide (ANP) against lung endothelial barrier dysfunction caused by edemagenic and inflammatory mediators. We discovered PKG-independent barrier protective effects of ANP in lung endothelium and demonstrated differential role of ANP type A and C receptors in the ANP anti-inflammatory effects. We also demonstrated an essential role of Rac1 GTPase-activated PAK1 kinase in the protective effects of ANP against vascular leak and inflammation induced by Gram-negative bacterial compounds. My most recent findings suggest that stathmin-dependent signaling mediates ANP protective effects against agonist-induced lung injury.

a. Xing J, Moldobaeva N, Birukova AA. Atrial natriuretic peptide protects against Staphylococcus aureus-induced lung injury and endothelial barrier dysfunction. J Appl Physiol, 2011;110(1):213-24.

b. Xing J, Yakubov B, Birukova AA. Opposite effects of ANP receptors in attenuation of LPS-induced endothelial permeability and lung injury. Microvasc Res,  2012;83(2):194-9.

c. Tian X, Tian Y, Gawlak G, Sarich N, Wu T, Birukova AA. Control of vascular permeability by atrial natriuretic peptide via GEF-H1-dependent mechanism. J Biol Chem. 2014 Feb 21;289(8):5168-83.

d. Tian Y, Mambetsariev I, Sarich N, Meng M, Birukova AA. Role of microtubules in attenuation of PepG-induced vascular endothelial dysfunction by atrial natriuretic peptide. Biochim Biophys Acta. 2015 Jan;1852(1):104-19.

Hepatocyte growth factor (HGF) is another natural molecule present in circulation and associated with the lung recovery in ALI/ARDS. We identified relations between HGF and cell ventilation-relevant mechanical stimulation of pulmonary EC in the control of cell cytoskeleton and barrier and reported the involvement of Rac-specific nucleotide exchange factor Tiam1 in the HGF-induced endothelial barrier enhancing effects. Our most recent studies showed that HGF-mediated Rac activation, cell junction enhancement, and preservation of EC barrier against agonist-induced hyperpermeability are tightly associated with signaling by MT.We also described the involvement of novel Rac-specific nucleotide exchange factor Asef in the mediation of HGF barrier-enhancing effects. We studied interplay between Tiam1 and Asef and found that MT control Asef, but not Tiam1 nucleotide exchange activity. Until now, the role of Asef in the regulation of endothelial barrier function remained unknown. Our studies demonstrate a key role of Asef in the HGF anti-inflammatory effects. Therefore, signaling by Asef is critical for both, preservation of vascular endothelial integrity, accelerated recovery and modulation of EC inflammation caused by bacterial pathogens. Modulation of Asef activity may have important implications in therapeutic approaches for the treatment of sepsis and ALI/ARDS.

a. Birukova AA, Alekseeva E, Mikaelyan A, Birukov KG. HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway. FASEB J, 2007, Sep;21(11):2776-86.

b. Higginbotham K, Tian Y, Gawlak G, Moldobaeva N, Shah A, Birukova AA. Hepatocyte growth factor triggers distinct mechanisms of Asef and Tiam1 activation to induce endothelial barrier enhancement. Cell Signal. 2014 Nov;26(11):2306-16.

c. Meng F, Meliton A, Moldobaeva N, Mutlu GM, Kawasaki Y, Akiyama T, Birukova AA. Asef mediates HGF protective effects against LPS-induced lung injury and endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol. 2015 Mar 1;308(5):L452-63.

d. Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA. Asef controls vascular endothelial permeability and barrier recovery in the lung. Mol Biol Cell. 2015 Feb 15;26(4):636-50.

4. Control of lung endothelial barrier by small GTPases and its regulation by guanine nucleotide exchange factors.  

Small GTPases Rac and Rho play opposing roles in EC cytoskeletal remodeling and cell junction dynamics, the critical mechanisms of endothelial barrier regulation. In contrast to the now recognized role of MT disassembly in activation of Rho pathway leading to EC barrier dysfunction, a role of MT-associated signaling in the regulation of barrier protective Rac signaling remains poorly understood. Our recent studies demonstrated that Asef binding to MT-associated protein adenomatous polyposis coli (APC) adaptor protein targets Asef to microtubules. Growing microtubules transmit Asef/APC complex to the cell periphery where it activates Rac and Rac effectorIQGAP1, and stimulates IQGAP1 interactions interactions with MT plus-end protein EB1 and actin binding protein cortactin. These data indicate that IQGAP1 functions as a hub, linking Rac signaling to MT and actin remodeling via EB1-IQGAP1-cortactin interactions. This represents a novel mechanism of local regulation of Rac and endothelial barrier function.

a. Tian X, Tian Y, Moldobaeva N, Sarich N, Birukova AA. Microtubule dynamics control HGF-induced lung endothelial barrier enhancement. PLoS One. 2014 Sep 8;9(9):e105912.

b. Tian Y, Tian X, Gawlak G, O'Donnel JO, Sacks DB, Birukova AA. IQGAP1 regulates endothelial barrier function via EB1 - cortactin crosstalk. Mol Cell Biol. 2014 Sep 15;34(18):3546-58.

c. Tian Y, Gawlak G, Shah AS, Higginbotham K, Tian X, Kawasaki Y, Akiyama T, Sacks DB, Birukova AA. HGF-induced Asef-IQGAP1 Complex Controls Cytoskeletal Remodeling and Endothelial Barrier.J Biol Chem. 2015 Feb 13;290(7):4097-109.

5. Current research projects.

- Microtubule control of septic inflammation: this study explores regulation of endothelial inflammation caused by Staphylococcus aureus pathogen via microtubule-associated signaling mechanisms.

Supported by R01 GM114171, 08/15/2015-05/31/2019

- Microtubule-associated regulation of acute lung injury: this study investigates a new mechanism of microtubule-dependent control of lung vascular dysfunction and acute lung injury caused by bacterial endotoxin.

Supported by R01 HL107920, 04/01/16-03/31/20

- Differential mechano-signaling in vascular endothelium by varying degrees of mechanical stretch: the goal of this project is studying of magnitude-dependent regulation of lung endothelial barrier recovery via mechanosensitive regulation of small GTPases at the cell junctions. Supported by R01 HL 130431, 07/01/16-04/30/20

Complete list of Published Work can be found at: http://www.ncbi.nlm.nih.gov/pubmed/?term=birukova