Krüppel-like Factor 5 contributes to pulmonary artery smooth muscle proliferation and resistance to apoptosis in human pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a vascular disease that is mainly restricted to small pulmonary arteries. PAH occurs in rare idiopathic and familial forms, but is more commonly part of syndromes associated with connective tissue diseases, anorexigen use, HIV or congenital heart disease. This syndrome of obstructed, constricted small pulmonary arteries (PA) has been attributed to abnormalities in the blood content of some neurotransmitters and cytokines, namely increases in serotonin, IL-6, PDGF and endothelin-1 [1‐4]. We recently demonstrated that the increase in these circulating vasoactive agents triggers in pulmonary artery smooth muscle cells (PASMC) the activation of the nuclear factor of activated T-cells (NFAT) contributing to increase [Ca²⁺]_i-mediated PASMC proliferation [5, 6]. Moreover, we showed a sustained increase in the oncoprotein survivin, decreasing mitochondrial-dependent apoptosis [7]. The fact that the PAH phenotype is preserved in cultured PASMC isolated from PAH patients suggests that the PAH phenotype is sustained independently of the circulating growth factors or agonists but requires genetic remodeling processes [8, 9]. Moreover, despite recent therapeutic advances such as endothelin-1 receptor blockers (e.g. bosentan) [10], type 5 phosphodiesterase inhibitors (e.g. sildenafil) [11] or PDGF receptor blockers (e.g. imanitib) [12], mortality rates remain high [13].

Krüppel-like factor 5 is a zinc finger transcription factor that belongs to a family known as the Sp/KLF factors, and is implicated in important biological functions including cell proliferation, apoptosis, development, and oncogenic processes [14‐16]. In Vascular Smooth Muscle Cells (VSMC) KLF5 regulates expression of the embryonic form of smooth muscle myosin heavy chain (SMemb/NMHC-B), which is selectively expressed in the proliferative dedifferentiated smooth muscle phenotype. In systemic vessels, KLF5 is expressed in proliferating smooth muscle cells of coronary artery lesions [17], and expression of this factor in lesions is clinically associated with restenosis and cardiac allograft vasculopathy [17]. KLF5 expression is therefore associated with proliferating smooth muscle cells in the cardiovasculature [18, 19]. However, it had yet to be shown whether KLF5 is activated in PAH-PASMC and whether it's implicated in PASMC proliferation and apoptosis.

All experiments were performed in accordance to the Université Laval's Ethic and Biosafety Committee (protocol number 20142) and the Centre Hospitalier Universitaire de Québec's Ethic's Committee.

Here we show that KLF5 is etiologically associated with the development of PAH, and believe that we have opened a new avenue for PAH treatment. KLF5 is expressed in established human and experimental PAH. Its expression is dependent of the activation of STAT3. We provided evidences that KLF5 inhibition improves PAH by 1) the inhibition of cyclin B1 and PASMC proliferation and 2) the depolarization of PASMC mitochondria by inhibiting survivin activation thus increasing apoptosis. These findings not only confirm what has been shown in cancer [16, 31, 32] and systemic vascular smooth muscle cells [33, 34] in which KLF5 inhibition decreases proliferation through cell cycle protein inhibition like cyclin B1 and p21 upregulation, but also provide a better demonstration of the involvement of KLF5 in mitochondrial-dependent apoptosis. In fact, we provide for the first time evidences that KLF5 is implicated in mitochondrial membrane potential regulation through the upregulation of the oncoprotein survivin. We have previously extensively demonstrated the mechanism of mitochondrial membrane potential regulation by survivin in PAH-PASMC [7], but had not elucidated the mechanism accounting for its upregulation. Our new findings propose that KLF5 activation might explain such upregulation. In fact, we showed that KLF5 inhibition decreases survivin expression in PAH-PASMC. This is associated with a significant mitochondrial depolarization. This finding could be of great therapeutic interest as it provides a new insight on the regulation of survivin that is implicated in many cancer and cardiovascular diseases [35‐39]. Thus, our findings might not be limited to PAH but can be extend to many other diseases including cancer. To this end, a recent report has demonstrated a link between KLF5 and survivin in cancer [40].

Previous studies reported that KLF5 is implicated in embryonic stem cells and VSMC differentiation contributing to vascular lesions [34, 41]. This aspect might be implicated in the vascular lesions seen in PAH patients. Indeed, vascular lesions such as remodeled arteries and plexiform lesions are seen in patients with PAH. Studies have demonstrated the implications of abnormal stem cells in this phenomenon [42]. KLF5 implication in such processes cannot be ruled out.

The activation of KLF5 axis that we described likely has a multifactorial etiology in PAH. However, KLF5 might be a critical integrator of multiple signaling pathways and its downstream effects might explain several and important features of PAH. In vivo, endothelial dysfunction is recognized as one of the earliest abnormalities in PAH, resulting in a well-recognized imbalance of endothelium-derived vasoactive factors; with increased vasoconstrictors (like endothelin [28], thromboxane [1] and decreased vasodilators (like NO or prostacyclin [1]). Recently, KLF5 has been shown to be implicated in endothelial dysfunction [43]. In addition, increased circulating growth factor (like PDGF) [27] and cytokines (like IL-6, MCP-1...) [26] have been reported in PAH, and there is numerous evidences demonstrating the implication of KLF5 in both growth factor and cytokines production regulation [18, 43‐45].

Finally, we have previously extensively demonstrated the implication of HIF-1 in triggering mitochondrial and [Ca²⁺]_i dysfunction sustaining the pro-proliferative and anti-apoptotic phenotype seen in PAH-PASMC [5, 46]. Interestingly, Mori et al. have shown that cooperation between HIF-1 and KLF5 might exist [47]. Indeed, KLF5 inhibition decreases the expression of several HIF-1-regulated genes in cancer cells, while HIF-1 inhibition affects KLF5 expression [47]. These findings in cancer cells implied that interaction between HIF-1/KLF5 in PAH-PASMC might exist. Thus, all these reports suggest that KLF5 might play a critical role in the etiology of PAH. Surprisingly no studies have been performed on the topic.

The mechanisms accounting for KLF5 upregulation in PAH remain to be established. Recently, a study from Cheng et al [48] has suggested the implication of the microRNA 145 (miR-145). In their study, they provided evidences that the downregulation of miR-145 promotes vascular neointimal lesion formation through the upregulation of KLF5. Nonetheless, downregulation of miR-145 in PAH, has not been demonstrated [49]. We believe that one of the initial events in PAH, regardless of the specific cause, is the activation of the miR-204/STAT3 axis [50], which increases KLF5 expression (Figure 2), supporting the notion that KLF5 induction is an early event during PAH. Thus, KLF5 upregulation can be caused by many diverse conditions that lead to PAH in addition to hypoxia, including growth factors, vasoactive molecules [50, 51], or viral infection of PASMC [52]. The fact that STAT3 activation may occur only in pulmonary, not systemic, vasculature [50] explains the selective induction of KLF5 in the pulmonary circulation. This finding strengthens the argument that KLF5 therapeutic targeting may achieve relative selectivity for the pulmonary circulation in PAH. Although many more experiments are needed in order to determine whether this regulation is direct or indirect, this finding is of great interest. In fact, STAT3 has been associated with many feature of PAH including BMPR2 downregulation (hallmark of PAH) [53]. Involvement of KLF5 in the STAT3-mediated effects in PAH will certainly open new avenues of investigation. Finally, we showed that both indirect (through STAT3 inhibition) and direct KLF5 inhibition (nebulized siRNA) improve PAH. Nonetheless, we should note that STAT3 inhibition showed a greater efficiency than siKLF5. This is not surprising as STAT3 has been implicated in many features characterizing PAH, including miRNA such as miR-204, Pim1; NFAT; BMPR2 [6, 50, 51, 53]

Although our findings will need to be repeated in greater amount of patients and at different stages of PAH to have a better understanding of the role of KLF5 in PAH, nonetheless our study suggests for the first time the implication of KLF5 in the etiology of human PAH. Moreover we provide evidences that its activation might account for many feature seen in PAH, including PASMC proliferation, mitochondrial hyperpolarization, survivin expression and resistance to apoptosis. Because, pulmonary arterial hypertension is a rapidly lethal disease for which treatments are limited, we believe that our findings will open the door to new avenues of investigation and potentially future therapies for PAH.