Effects of MCT Treatment

Using oligonucleotides spotted glass chips, we characterized the expression pattern in the lung tissue obtained from the three different groups i.e. MCT, MCT+Tola (MT) and Tola alone to compare with that found in normal lung tissue. So far, less work has been performed in context to identified candidate genes that are involving in PH disease.

Recently, Norbert F. Voelkel et al.investigated differentially regulated genes in PPH human samples.⁹⁷ Here, for selecting differentially regulated genes in each groups, we applied a regulation criterion that is a twofold change in expression [log2 ratio of 1.0 (upregulation) or -1.0 (downregulation)].

As the inflammatory mechanisms appear to play a significant role in MCT-PH model,³⁶ we hypothesized that factors such as inflammatory mediators, proteases and elastase, matrix metalloproteinase, ion channels, or vascular endothelial growth factors may have role in progression of the disease. We obtained 103 genes, which were regulating with a 2-foldchange. Few of them are discussed here.

A link between MCT-induced inflammation andsmooth muscle cell (SMC) proliferation was first suggested bycertain findings in pulmonary hypertension. SMCproliferation has also been demonstrated in the pulmonary vasculature in chronic PH of animal models.⁹⁸ Proinflammatory interleukins are intriguing candidates for a MCT-induced SMC-derived autocrine growthfactor. As expected, pro-inflammatory genes like IL-3 receptor beta and IL-1 receptor 1, excessively produced in the lungs of rats treated with monocrotaline,⁹⁹ were upregulated here. Overexpressions of these cytokines stimulate proliferation of SMC. Such proliferation may contribute to the pathophysiological effectsof chronic MCT induced PH and thus may play a rolein the development of pulmonary hypertension.

Overexpression of arachidonate 5-lipoxygenase and chemoattractant protein-1 (MCP-1) was shown in endothelial cells of plexiform lesions and inflammatory cells in patients with PPH.¹⁰⁰ Interestingly, these genes were upregulated with more than a factor of 2.0, suggesting that upregulation of these enzymes involved in generation of inflammatory mediators and may play a role in the pathogenesis of PH.

Mast cells are specialized immune effector cells that synthesize and store in their granules large amounts of serine proteases, marked, statistically significant increases in proteases (particularly derived from mast cells) of vascular obliteration and pulmonary hypertension. Mast-cell-derived proteases may play a role in regulating the development of neointimal pulmonary vascular occlusion and pulmonary hypertension in response to injury. Indicating involvement in the remodelling process, our array data show that several mast-cell-derived proteases like mast-cell protease 1, 5, 7 and 8 precursors were highly upregulated by more than a factor of 5.0. Similar regulation pattern for mast cell proteases in MCT rat model was reported by Laszlo T et al.¹⁰¹ In addition to this, other proteases like serine protease was found to be upregulated.

Increased activity of serine proteases in pulmonary arteries has been observed during early remodelling in MCT-induced pulmonary hypertension.¹⁰²

Besides mast cell proteases and serine proteases, some other proteases expression was found to be significantly elevated during the development of disease, for example, cathepsin K (3.0-fold) and cathepsin Y (approx. 2.0-fold) which has an influence on developing PH. Cathepsin K is a protease with potent fibrinolytic activity that may play an important role in extracellular matrix degradation. Again Laszlo T et al. showed upregulation of cathepsin K in MCT rat model.

Important changes occur in PH in the vascular adventitia, with increased production of the extracellular matrix. Cowan et al. recently showed that direct inhibition of serine elastases led to complete regression of pathological changes in experimental PH caused by monocrotaline.¹⁰³ Because metalloproteinases are potentially destructive agents, their production is tightlycontrolled at several levels. A way in which the activity of MMPsis regulated is based on the presence and activity of specific inhibitors such as TIMP-1,^104-105 which was upregulated, such overexpression is due to the interaction of MMPs with their specific inhibitors that determines the net activity of secreted enzymes during pulmonary artery remodelling. Because our results showing that mast cell proteases, cathepsins, and TIMP are increased during progression of pulmonary hypertension that extends the observations that experimental pulmonary hypertension involves extensive remodelling of extracellular matrix. ¹⁰³

Upregulation of Insulin-like growth factor-1 IGF has been recognized as likely having a role in stimulating smooth muscle hyperplasia.¹⁰⁶ IGF-I directly stimulate fibroblast

proliferation and perhaps collagen synthesis.¹⁰⁷ We found upregulation of IGF, such overexpression of IGF-1 can leads to increased cellproliferation decreased apoptosis and increased smooth muscle elastin synthesis that may play an important role in vascular remodelling in chronic pulmonary hypertension.

A member of the metal binding protein family is metallothionein 2 (MT-2), a low–

molecular weight, cysteine-rich, heavy metal-binding protein can be easily induced by heavy metals, hormones, acute stress, and various chemicals.¹⁰⁸ In our approach, MT-2 was upregulated. This enzyme might play a role in the pulmonary defense against zinc toxicity and free radicals. As MCT rat model is a chronic PH model it influences upregulation of MT-2 against toxicity and free radicals. Interestingly, upregulation of metallothionein 1 and 2 in pulmonary arteries were found in parallel during hypoxia induced pulmonary hypertension (unpublished data communicated by L. Fink).

Furthermore, MT-2 previously has been shown to increase after an ischemic insult.¹⁰⁹ Flavin-containing monooxygenase 1 (FMO) is a member of xenobiotic-metabolizing enzymes family. In normal condition, FMO oxidatively metabolizes a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics compounds. In our setting, downregulation of FMO indicates the increased deposition of toxic reactive intermediates that might involve in a vascular injury.

On the other hand several genes were differentially downregulated in response to MCT. Among downregulated genes most of them are belongs to kinases family, for example; fms-related tyrosine kinase 1, protein kinase c, serine-threonine kinase receptor type I activin, and Inositol hexakisphosphate kinase (IP6) enzymes were found to be downreguled. Kinases have been shown in vascular smooth muscle cells to regulate the expression of p27KIP1 leading to an enhanced G1/S transition of vascular smooth muscle cells.¹¹⁰ A member of apoptosis regulation family i.e. IP₆ in response to MCT was downregulated. Members of this family involved in apoptosis regulation.¹¹¹ Furthermore, IP₆ is a naturally occurring polyphosphorylated carbohydrate that is present in substantial amounts in almostall mammalian cells, which are important for regulating vital cellular functions. It was recently recognized to possess multiple biological functions. A striking anticancereffect of IP₆ was demonstrated in different experimental models IP₆ is involved in apoptosis and vessel maintenance.¹¹²

Some receptors play a key role in angiogenesis. Ephrin b1 was downregulated and is known to be involved in vascular development and angiogenesis.¹¹³ Downregulation of ephrin b1 results in reduced vessels sprouting and development.

Interestingly, the most downregulated gene in our approach is BMPR-II that is known to have an important role in PPH. Diverse mutations in BMPR-II receptor followed by functional loss of BMPR II have been reported in patients with PPH.¹¹⁴ BMPR2 encodes a type II receptor member of the transforming growth factor-ß (TGF-ß) superfamily. Type II receptors have serine/threonine kinase activity and act as cell-signallimg molecules. Following ligand binding, type II receptors form heterodimeric complexes with membrane-bound type I receptors. This initiates phosphorylation of the type I receptor and downstream signalling via intracellular Smads.¹¹⁵ This pathway is diverse and the specificity in cell growth and differentiation is mediated through transcriptional control. Thus, for the first time downregulation of BMPR2 was found in an animal model of PH.