Pulmonary Hypertension Associated With Scleroderma and Connective Tissue Disease: Potential Molecular and Cellular Targets

Agonistic Anti-AT1R and Anti-ETAR Antibodies

Increased activation of vasoactive mediators angiotensin II (Ang II) and endothelin-1 (ET-1) has been reported in SSc and implicated in both vasculopathy and fibrosis.89 Angiotensin-converting enzyme (ACE) inhibitors, angiotensin 1 receptor (AT1R) antagonists, as well as ET-1 receptor blockers are routinely used to alleviate some of the vascular complications of SSc.10 Importantly, functional pathogenic antibodies directed against AT1R and endothelin-1 type A receptors (ETAR) were identified in patients with SSc and linked to increased prevalence of SSc-related vascular and fibrotic complications and higher risk for SSc-induced mortality.1112 Studies have shown that those antibodies activate respective surface receptors on endothelial cells and fibroblasts and exert proinflammatory and profibrotic effects.13 Recent studies by Becker et al have demonstrated higher levels of anti-ETAR and anti-AT1R antibodies in patients with SSc-PAH and connective tissue disease (CTD)-PAH compared to idiopathic pulmonary arterial hypertension (IPAH) and other forms of pulmonary hypertension (PH).11 Concomitant presence of anti-ETAR and anti-AT1R antibodies in patients with SSc predicted development of PAH and its associated mortality. In functional studies, injections of antibody-positive human SSc-immunoglobulin G (IgG) into mice induced obliterative vasculopathy with lymphocyte infiltration, as well as increased alpha-smooth muscle actin (αSMA) expression around the wall of small pulmonary vessels; however, Fulton's index and pulmonary vascular resistance (PVR) were not different from controls.11 Although findings from those studies suggest that the presence of antibodies alone is not sufficient to induce PAH, they nonetheless strongly support the pathogenic role of anti-ETAR and anti-AT1R antibodies in SSc-PAH and CTD-PAH patients.

Stimulatory Anti-PDGF Receptor Antibodies

Platelet-derived growth factor (PDGF) is one of the key growth factors involved in the pathogenesis of SSc.1415 PDGF is also involved in the pathogenic vascular alterations in PAH.16 Gabrielli and colleagues described agonistic pathogenic anti-PDGF receptor antibodies in SSc patients.17 These autoantibodies functioned similarly to PDGF in inducing intracellular signaling and reactive oxygen species (ROS) production.17 Furthermore, unlike natural ligands that elicit short-term effects, the effects of the anti-PDGF receptor autoantibodies were long lasting. With respect to PH, recent work from Svegliati el al demonstrated that autoantibodies targeting PDGF receptors induce proliferation and collagen synthesis, as well as NOX4 and ROS production in human pulmonary artery smooth muscle cells (HPASMC).18 Furthermore, these effects were, in part, mediated through the activation of MTORC1. Although clinical association of anti-PDGF receptor antibodies with SSc-PAH have not yet been examined, the latter studies suggest that stimulatory antibodies targeting PDGF receptors could activate vascular smooth muscle cells and, thus, contribute to the development of vascular lesions.

Other Autoantibodies Associated With SSc-PAH

Anti-endothelial cell antibodies (AECA) are detected in a large proportion of patients with SSc and are associated with vascular manifestations, including PAH.19 Cellular targets of AECAs have recently been characterized using proteomic approaches and include ubiquitous cellular proteins such as lamin A/C, tubulin, and vinculin.20 The presence of antifibroblast antibodies (AFA) has been described by several groups in patients with SSc-PAH.21–23 Those antibodies recognize many cellular targets; however, their functional role in disease remains to be investigated. Bussone et al reported increased frequency of antibodies to vascular smooth muscle cells (VSMC) in patients with SSc-PAH compared to IPAH and healthy controls.24 The authors also showed that serum IgG from those patients induced contraction of VSMCs in a collagen matrix, suggesting a pathogenic role. Given the widespread presence of self-reactive IgG autoantibodies in human sera, it may be expected that many additional functional autoantibodies will be characterized in SSc patients in the future.7

Inflammation

Animal models of PAH and clinical data support a role for perivascular inflammation in the pathogenesis of PAH.25 Because human diseased lung tissue is not readily available, global gene expression has been analyzed by several investigators in SSc-PAH peripheral blood mononuclear cells (PBMC).26–29 In general, those studies found elevated expression of inflammatory and vascular injury-related genes in SSc-PAH PBMCs compared to controls, although there was little overlap between the genes identified in the individual studies.

Increasing evidence supports the role of leukotrienes (LT), and, in particular, leukotriene B₄ (LTB₄), in the pathogenesis of PAH.30 LTB₄, which is produced by perivascular macrophages, induced endothelial cell apoptosis by inhibiting expression and activation of sphingosine kinase and endothelial nitric oxide synthase (eNOS). LTB₄ also induced proliferation and hypertrophy of human pulmonary arterioles smooth muscle cells (PASMC). Furthermore, LTB₄ enhanced proliferation, migration, and myofibroblast differentiation of adventitial fibroblasts. The serum concentration of LTB₄ was found to be significantly increased in PAH, primarily in patients with SSc-PAH.31

Associations of other serum biomarkers with SSc-PAH have been reported. For example, elevated levels of pentraxin 3 were found in patients with digital ulcers and PAH.32 A recent study revealed elevated levels of adipsin in lcSSc patients, which correlated with significantly increased risk for PAH and related cardiac dysfunction.33 Adipsin, a serine protease produced by adipocytes, also known as a complement factor D, has not previously been associated with rheumatic diseases. Whether adipsin plays a pathogenic role in SSc-PAH remains to be determined.

Perivascular Inflammation

In PAH, immune and inflammatory cells reside primarily in the outer layer of the vessel wall, the adventitia. Vascular adventitia is composed of various cells, including fibroblasts, blood and lymphatic vessels, nerve cells, as well as immune cells embedded in the connective tissue.34 Comprehensive comparison of the immune cells in control and IPAH lungs was recently reported by Savai et al.35 The study showed a significant increase in monocytes and macrophages (CD14⁺ and CD68⁺), dendritic cells (CD209⁺), T cells (CD8⁺ and CD4⁺) in IPAH lungs, as compared to controls. Notably, the immune cells were primarily localized to the adventitia. However, Foxp3⁺ cells were significantly decreased, consistent with a reduced presence of regulatory T cells.

Of particular relevance to the pathogenesis of PAH are macrophages, a functionally heterogenous cell population, originally classified into M1 (classically activated, proinflammatory) and M2 (alternatively activated, anti-inflammatory) subsets.36 It is now recognized that this classification does not adequately describe the spectrum of macrophage phenotypes, especially as it relates to the various disease conditions, including PAH. Studies have shown that in IPAH, as well as several experimental models of PH, adventitial macrophages were alternatively activated by interleukin (IL)-6 produced by the adventitial fibroblasts.37 At the molecular level, macrophages activated in this fashion showed increased signal transducer and activator of transcription 3 (STAT3), hypoxia-inducible factor 1-alpha (HIF1α), and CCAAT/enhancer binding protein beta (C/EBPβ) signaling, as well as higher expression of arginase and other genes consistent with the phenotype of macrophages isolated from PH lesions.37 This mode of activation differs from the established alternative macrophage activation pathways such as IL-4/IL-13/signal transducer and activator of transcription 6 (STAT6) or toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88). Fibroblast-activated macrophages were also metabolically reprogrammed to aerobic glycolysis, an altered tricarboxylic acid (TCA) cycle, and reduced mitochondrial respiration, discussed below.

Macrophages play an essential role in the pathogenesis of PH and their depletion mitigates pulmonary vascular remodeling.38 A recent study using hypoxia-induced PH in mice showed that M2 macrophages can be therapeutically targeted.39 Global deletion of a chemokine receptor CX3CR1 or treatment with pharmacological inhibitor of CX3CR1 led to significant reduction of PH with concomitant decrease of M2 and increase of M1 markers, suggesting a role for CX3CR1 as a therapeutic target in PAH.

Genetic Factors

Mutations in the components of the transforming growth factor beta (TGFβ) superfamily, including bone morphogenetic protein receptor-2 (BMPR2), activin-receptor-like type I (ACVRL1), endoglin (ENG), SMAD8, as well as caveolin-1 (CAV1) have been identified in hereditary forms of PAH and in the subset of IPAH patients.40 Although activation of TGFβ signaling is considered central to the pathogenesis of SSc,41 to date only the association between polymorphism in the ENG gene and SSc-PAH has been reported.42 Genetic variations in several other genes, including TLR2, tumor necrosis factor alpha-induced protein (TNFAIP) 3, and urokinase plasminogen activator (uPAR) have been associated with SSc vascular complications, as well as PAH.43–45 In addition, HLA-B*35 (human leukocyte antigen class B) has been shown to be associated with increased risk for developing PAH in Italian SSc patients4647 and in Brazilian SSc patients48; however, it is unclear whether this correlation is present in other SSc cohorts. Functional studies in endothelial cells have demonstrated that the presence of HLA-B*35 contributes to endothelial cell dysfunction by increasing production of ET-1 and significantly decreasing nitric oxide synthase in conjunction with upregulation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR).4950 The association of HLA-B*35 with increased expression of ER stress markers and inflammatory genes was further verified in PBMCs obtained from SSc patients.51

Endothelial to Mesenchymal Transition

Endothelial to mesenchymal transition (EndoMT) is a process whereby endothelial cells undergo molecular changes that result in a gradual loss of endothelial cell characteristics and emergence of mesenchymal cell markers. A number of studies using various experimental models of fibrosis have implicated EndoMT as a potential source of fibroblasts/myofibroblasts.6061 The presence of EndoMT has been demonstrated in human PAH and in 2 experimental models of PAH.62 Similar observations were also made in SSc-PAH. Good et al demonstrated the presence of cells coexpressing von Willebrand factor (vWF) and αSMA in pulmonary vessels of patients with SSc-PAH.63 EndoMT may also contribute to the fibrotic process in SSc-associated pulmonary fibrosis.64 Likewise, coexpression of endothelial cell markers cluster of differentiation (CD31) or vascular endothelin cadherin (VE-cad) and αSMA was frequently observed in SSc dermal capillaries and arterioles.65 Further in vitro comparison of SSc and healthy control microvascular endothelial cells showed higher expression of mesenchymal markers, such as collagen, αSMA, and S100 calcium-binding protein A4 (S100A4)/fibroblast-specific protein-1 (FSP1) and lower expression of CD31 and VE-cad in SSc endothelial cells. Interestingly, sera from SSc patients but not healthy controls induced EndoMT in control endothelial cells with a potency similar to that of TGFβ, suggesting that a factor(s) in SSc sera is contributing to the EndoMT in vivo. Inflammatory cytokines have been implicated in inducing EndoMT, suggesting that perivascular inflammation may contribute to this process in PAH.61 EndoMT appears to represent a common manifestation of vascular dysfunction; however, it is not yet clear whether endothelial cells serve as a primary source of αSMA expressing cells in the pulmonary lesions.

Transcriptional Regulation of Vascular Cells

Dysregulated transcription factor network has been associated with altered behavior of vascular smooth muscle and endothelial cells in PAH.66 Recent studies have demonstrated that the level and activity of one of the key regulators of VSMC proliferation and apoptosis, transcription factor forkhead box protein O-1 (FoxO1), is reduced in VSMCs from IPAH patients, as well as 2 experimental models of PH.67 Genetic or pharmacological inhibition of FoxO1 in VSMCs recapitulated PH characteristics in vivo, while re-expression of FoxO1 reversed the development of PAH and restored normal lung and heart function. Thus, FoxO1 deficiency in VSMCs appears to play a key role in the development of PH and its restoration may have a beneficial therapeutic effect. Additional transcription factors implicated in pro-proliferative and anti-apoptotic behavior of smooth muscle cells include Krüppel-like factor 5 (KLF5) and Oct-4.6869 Both factors are expressed at elevated levels in PASMCs isolated from IPAH patients, while elevated KLF5 expression was also confirmed in IPAH lung biopsies. Functional studies have shown reduced proliferation and increased apoptosis in response to KLF5 inhibition in PAH-PASMCs.68 Furthermore, in vivo inhibition of KLF5 improved PAH in a monocrotaline-induced PAH in rats.

Pulmonary veno-occlusive disease (PVOD) is characterized by the preferential involvement of the pulmonary venous system resulting in progressive elevation in pulmonary arterial pressure, leading to right heart failure and death.70 PVOD appears to be relatively common in patients with CTD and my affect up to 75% of patients.71 Heritable PVOD is caused by biallelic mutation in EIF2AK4 (eukaryotic initiation translation factor 2 alpha kinase 4) gene.70 EIF2AK4, also known as GCN2, is 1 of 4 kinases responsible for phosphorylating eukaryotic initiation phosphorylation factor 2 (eIF2), a key regulator of cellular response to various forms of stress. Studies have shown that GCN2 protects against hepatoxicity during aspariginase treatment, while the loss of GCN2 promotes oxidative stress and inflammation-mediated DNA damage.72 The role of GCN2 deficiency in the development of PVOD awaits further investigation.

Recent studies have linked suppression of the erythroblast transformation-specific (ETS)-related gene (ERG) to PVOD.73 ERG, a member of the ETS family of transcription factors, is involved in several developmental processes, including endothelial cell specification and blood vessel formation.74 ERG is highly expressed in differentiated endothelial cells, including large arterial, venous, and microvascular endothelium. Lathen et al have demonstrated that protein levels of ERG were significantly decreased in patients with PVOD.73 They further showed that ERG regulates vein-specific expression of Apelin receptor (APLNR). Mice with the homozygous deletion of ERG or APLNR developed lethal PVOD. These studies support the key role for the ERG-APLNR axis in maintaining pulmonary venous endothelial homeostasis. The potential interactions between ERG-APLNR and GCN2 in the pathogenesis of PVOD remain to be established. Interestingly, recent studies have shown that ERG could also be involved in regulating inflammation in pulmonary vasculature.75 Looney et al showed that endothelial ERG protein expression was reduced in patients with IPAH and SSc-PAH, as well as in the lungs of hypoxic mice.75 Loss of ERG in the pulmonary endothelial cells led to de-repression of interferon beta 1 (IFNβ-1) gene and marked upregulation of interferon-responsive proinflammatory genes. Friend leukemia integration factor 1 (FLI1) is a close homolog of ERG and, like ERG, is involved in regulation of vascular stability.76 Simultaneous depletion of ERG and FLI1 had a synergistic or additive effect on expression of the inflammatory genes. Furthermore, ERG and FLI1 double-heterozygous mice showed increased inflammation in the lung.75 It has been proposed that ERG and FLI1 may represent the “master” transcription factors regulating the crucial manifestation of PH such as inflammation and small artery remodeling.77 Further support for the central role of ERG in regulating vascular inflammation came from the work of Hogan et al.78 Using a combination of computational and experimental approaches the authors identified activator protein 1 (AP1), ETS, and GATA family members as central transcriptional regulators of endothelial cell homeostasis. Among the multiple members of those families, JUN, JUNB, JUND, and ERG were highly expressed in human aortic endothelial cells. Consistent with other studies, knockdown of ERG in human aortic endothelial cells elicited proinflammatory gene expression.78

There is also evidence that dysregulated expression of GATA-6 may contribute to PAH. It has been reported that GATA-6 protein levels are markedly reduced in pulmonary endothelial cells and smooth muscle cells in patients with IPAH and SSc-PAH.79 GATA-6 is a direct transcriptional regulator of genes controlling vascular tone, including ET-1, ETAR, and eNOS, as well as CX-3CL1. Mice with endothelial deletion of GATA-6 (GATA-6 knockout [KO]) spontaneously develop elevated pulmonary pressure and right ventricular hypertrophy.79 After exposure to hypoxia, GATA-6 KO mice display marked worsening in pulmonary pressure that is paralleled by extensive vascular remodeling.

Metabolism and Mitochondrial Dynamics

In recent years, it has been recognized that there are striking similarities between phenotypic changes occurring in PAH vasculature and cancer.80 These findings gave rise to the metabolic theory of PAH.81 Like cancer cells, PAH-PASMCs and endothelial cells are hyperproliferative and resistant to apoptosis.81–83 Furthermore, akin to cancer cells, vascular cells in PAH undergo a metabolic switch from mitochondrial oxidative phosphorylation to aerobic glycolysis. Mitochondrial alterations, which are both structural and functional, have been primarily investigated in PASMCs. PH-PASMCs have hyperpolarized mitochondria that produce less ROS. The changes in the mitochondrial metabolic redox signaling are associated with activation of HIF1 and nuclear factor of activated T cells (NFAT). Once activated, HIF1 upregulates transcription of most of the glycolytic enzymes. Furthermore, mitochondrial pyruvate dehydrogenase complex (PDH), a key enzyme in glucose metabolism that links glycolysis to the TCA cycle, is inactivated by pyruvate dehydrogenase kinase (PDK), also a target of HIF1. Both HIF1 and NFAT contribute to the downregulation of Kv channel expression, a well-known feature of PAH. Structural alterations of mitochondria include fragmented mitochondrial network due to the imbalance of mitochondrial biogenesis characterized by dysregulated expression of mitochondrial fission and fusion genes.83

PH fibroblasts also exhibit a hyperproliferative and inflammatory phenotype that is caused by metabolic reprogramming.8284 The cellular basis underlying these changes was recently elucidated by Stenmark and colleagues. They found a switch of pyruvate kinase isoenzymes PKM1 to PKM2 in PH fibroblasts. PKM1/2 is an enzyme that catalyzes the last step of glycolysis in which pyruvate and adenosine triphosphate (ATP) are formed. A switch to a less active PKM2 isoform leads to elevated glycolytic reprogramming and facilitates generation of macromolecule precursors that are necessary for rapid cell proliferation.82 Of note, PKM2, which also has many nonmetabolic functions, is widely overexpressed in tumors and supports tumor cell growth.85 Metabolic reprogramming toward aerobic glycolysis in PH fibroblasts led to enhanced levels of free reduced nicotinamide adenine dinucleotide (NADH), which in turn upregulated a transcriptional co-repressor C-terminal binding protein 1 (CtBP1).84 As a negative regulator of several tumor suppressor genes, CtBP contributes to proliferation and resistance to apoptosis in both tumor cells and PH fibroblasts.

Numerous studies support the role of excessive oxidative stress in the pathogenesis of SSc.8687 Fibroblasts isolated from SSc lesions spontaneously produce increased amounts of ROS.88 Likewise, circulating monocytes from SSc patients release more superoxide than controls.89 In addition, sera obtained from SSc patients but not from healthy controls induce ROS production in cultured fibroblasts and endothelial cells.90 Oxidative stress has also been implicated in the development of SSc-PAH; for example, SSc-PAH patients have lower levels of exhaled nitric oxide (NO),9192 as well as elevated plasma levels of asymmetrical dimethylarginine (ADMA), which may further contribute to lower NO production.93 SSc PBMCs express significantly higher levels of genes representing all 3 branches of the UPR, including immunoglobulin heavy-chain binding protein (BiP), activating transcription factor (ATF) 6, ATF4, spliced X-box binding protein 1 (XBP1), and chaperone DNAJB1.29 Interestingly, there was a significant correlation between pulmonary arterial pressure and DNAJB1, as well as between IL-6 expression and BiP, suggesting that chronic UPR may contribute to increased inflammation in SSc. Analysis of plasma obtained from SSc patients with and without PAH revealed unfavorable metabolic profile characterized by deficiency of metabolites with protective effects on endothelial cells.94

PAH Therapies: Present and Future

Rapid progress in the understanding of the pathogenesis of PAH led to development of specific therapies targeting deficiencies in either NO signaling (phosphodiesterase type-5 inhibition or soluble guanylyl cyclase agonism), PGI2 (prostacyclin analogs), or excessive ET-1 (ET receptor blocker).95 Furthermore, a double or triple combination therapy offered benefits to patients with severe PAH.95 Despite these promising treatment options, the disease remains poorly managed, especially in SSc patients. In the final stages of PAH, lung transplant is the only option and, therefore, there is a significant need for alternative treatment options. The complexity of PAH, including impaired redox homeostasis, abnormal vascular remodeling, and immune cell activation suggests that a therapeutic agent capable of modulating several key pathways would be an attractive addition to the treatment regimen of PAH. The transcriptional regulator nuclear factor (erythroid-derived 2)-related factor 2 (NRF2) is a key regulator of the antioxidant genes.96 Importantly, NRF2 function is linked to nuclear factor kappa beta (NFκB) signaling with activation leading to an anti-inflammatory response. In support of NRF2-based therapy in PAH, bardoxolone methyl (CDDO-Me), an NRF2 inducer, has shown encouraging results in a phase 2 study in PAH.97 Dimethyl fumarate (DMF, BG-12,Tecfidera^®), another inducer of NRF2, has recently been approved in the United States for the treatment of multiple sclerosis (MS).98 DMF has an excellent safety record in MS and 2 decades of use in psoriasis, also an inflammatory condition. DMF has been evaluated in experimental models of PH99 and mitigated PH by targeting multiple signaling pathways including oxidative damage, inflammation, and fibrosis. Efforts are underway to conduct a pilot randomized controlled trial with Tecfidera in SSc-PAH.100