PI3Kγ inhibitory peptides for the treatment of fibroproliferative vascular disease

Abstract

A fusion peptide and pharmaceutical compositions comprising the same for use in treating, preventing, and / or delaying the onset of fibroproliferative vascular diseases, wherein the fusion peptide comprises: (a) an amino acid sequence as defined in SEQ ID NO: 1 or a related homologue having at least 85% similarity to SEQ ID NO: 1 and having the ability of SEQ ID NO: 1 to inhibit the kinase-independent function of PI3Kγ; and (b) Peptides with cell-penetrating ability Includes:

Description

[Technical Field]

[0001] This description concerns novel strategies for the therapeutic management of fibroproliferative vascular diseases. [Background technology]

[0002] Cardiovascular diseases account for the majority of morbidity and mortality worldwide. These pathologies are often due to vascular dysfunction and occlusion, and can occur even after interventional strategies used in the treatment of atherosclerosis. For example, intimal hyperplasia (IH) arises from the progression of atherosclerotic lesions, but can also occur after transvascular angioplasty and stent placement, which are standard treatments for atherosclerosis. IH exists in a complex multifactorial response of the vascular wall, leading to the formation of a multicellular layer within the arterial lumen. IH involves inflammatory and fibrous phenomena resulting from endothelial damage and subsequent dedifferentiation of vascular smooth muscle cells (VSMCs), leading to excessive proliferation and migration of VSMCs. Together, these processes can have dramatic consequences, ultimately leading to intimal thickening and decreased blood flow. Therefore, understanding the mechanisms of VSMC proliferation is crucial for developing novel therapeutic strategies to prevent IH [1, 2].

[0003] Among the signaling molecules involved in VSMC proliferation, the second messenger 3'-5' cyclic adenosine monophosphate (cAMP) plays a major role. In VSMCs, elevated cAMP levels inhibit mitogenically stimulated proliferation both in vitro and in vivo [3-6]. Consistently, cAMP has been found to have a positive role in reducing intimal thickening in several models of vascular injury. Furthermore, cAMP signaling is affected in a wide variety of vascular pathologies, including atherosclerosis or restenosis after angioplasty. cAMP signaling relies primarily on its local production by adenylyl cyclase (AC) and its degradation by cyclic nucleotide phosphodiesterase (PDE) [7]. In VSMCs, PDE3A, PDE4, and PDE1C are involved in regulating VSMC proliferation and IH [3, 8, 9]. Interestingly, cAMP production has been shown to be associated with increased PDE4 activity and upregulation of PDE4D isoform expression in both contractile and synthetic phenotypes

[10] . Consistently, a great many studies have been conducted using PDE inhibitors in various arterial disease models [3].

[0004] In this context, phosphoinositide 3-kinase γ (PI3Kγ), a lipid kinase involved in immunomodulation of cardiovascular disease, has been shown to regulate cAMP levels in cardiomyocytes via a kinase-independent mechanism. On the one hand, PI3Kγ can produce 3-phosphoinositide lipid second messengers as a kinase, and on the other hand, within the same macromolecular complex, it can act as an "A-kinase anchor protein" (AKAP) associated with cAMP-dependent kinases, also known as protein kinase A (PKA), and phosphodiesterases 3 and 4 (PDE3 and PDE4), which are cAMP-degrading enzymes regulated by PKA. Consistent with this view, the catalytic subunit of the PI3Kγ holoenzyme (p110γ) interacts with PKA, participating in a negative feedback loop that leads to the activation of PDE3 and PDE4, ultimately increasing cAMP degradation.

[0005] In cardiac cells, PI3Kγ suppresses the increase in cAMP in response to β-adrenergic stimulation, ultimately reducing myocardial contractility

[11] . In contrast, PI3Kγ-mediated regulation of cAMP levels in airway smooth muscle cells promotes contraction and leads to bronchoconstriction. Therefore, inhibition of the kinase-independent function of PI3Kγ in airway smooth muscle using cell-permeable competitive peptides promotes bronchoconstriction

[12] . [Overview of the project] [Problems that the invention aims to solve]

[0006] The purpose of this disclosure is to provide novel strategies for the treatment, prevention, and delay of fibroproliferative vascular disease. [Means for solving the problem]

[0007] According to the present invention, the above objectives can be achieved thanks to the gist of the following claims, which are understood to form an integral part of this disclosure.

[0008] The present invention relates to a fusion peptide and a pharmaceutical composition comprising a fusion peptide for use in the treatment, prevention, and / or delay of the onset of fibroproliferative vascular disease, wherein the fusion peptide is (a) an amino acid sequence defined in SEQ ID NO: 1 or an associated homolog having at least 85%, preferably at least 90%, more preferably at least 95%, identity with SEQ ID NO: 1 and having the ability to inhibit the kinase-independent function of PI3Kγ, and (b) Peptides that have the ability to permeate cells Includes.

[0009] Arterial remodeling observed in hypertension and intimal hyperplasia is accompanied by inflammation and impaired flow, both contributing to the dedifferentiation and proliferation of smooth muscle cells. In this application, we have for the first time identified the kinase-independent crucial role of non-hematopoietic phosphoinositide 3-kinase γ (PI3Kγ) in the vascular wall during intimal hyperplasia, using mouse models lacking PI3Kγ or expressing a kinase-dead version of the enzyme. Furthermore, we found that the absence of PI3Kγ in VSMCs results in increased intracellular cAMP levels and regulation of cell proliferation associated with VSMCs. Real-time analysis of cAMP dynamics reveals that PI3Kγ regulates cAMP degradation in primary VSMCs independently of its kinase activity, via the regulation of the phosphodiesterase (PDE) 4 enzyme. The present invention therefore relates to the therapeutic application of an N-terminal competitive peptide of PI3Kγ (having the amino acid sequence shown in SEQ ID NO: 1) or a corresponding homolog capable of blocking the proliferation of primary VSMCs. These data provide evidence for the kinase-independent role of PI3Kγ in arterial remodeling and reveal novel strategies targeting the docking function of PI3Kγ to treat / prevent / delay the onset of fibroproliferative vascular disease.

[0010] The present invention is described herein in detail with respect to the accompanying drawings, by purely illustrative and non-limiting embodiments. [Brief explanation of the drawing]

[0011] [Figure 1]PI3Kγ controls IH and VSMC proliferation. A. Representative cross-sections of injured femoral arteries stained with Masson Trichrome from the indicated hematopoietic chimeras. Scale bar = 100 μM. Irradiated WT, PI3Kγ KD (KD), and PI3Kγ KO recipient mice were transplanted with the indicated donor-derived bone marrow to obtain the indicated chimeras. Irradiated WT mice were transplanted with bone marrow from WT, PI3Kγ KD, or PI3Kγ KO to obtain the following hematopoietic chimeras: WT>WT, KD>WT, and KO>WT. Irradiated KD recipient mice were transplanted with bone marrow from WT or PI3Kγ KD donor mice (WT>KD; KD>KD). Irradiated KO recipient mice were transplanted with bone marrow from WT or KO donors (WT>KO and KO>KO). B. Quantitative analysis of the intima / media ratio of the indicated hematopoietic chimeras. Histograms represent the neointima / media ratio for each chimera (n > 6 mice for each group). Data are represented as mean ± SEM and were compared using one-way ANOVA test. C. Schematic of the experimental model used to investigate the involvement of PI3Kγ in the control of SMC proliferation. D. Western blot of PI3Kγ expression in each genotype. E. Proliferation rate expressed as the doubling rate measured by BrdU incorporation and compared to control of primary VSMCs from WT, PI3Kγ KO, or PI3Kγ KD aorta, incubated in blocking medium for 24 hours or treated with 25 ng / ml PDGF with or without the addition of 25 μM forskolin (5 < n < 12 cultures for each genotype). Data are represented as mean ± SEM and were compared using one-way ANOVA test. [Figure 2]In VSMCs, PI3Kγ regulates cAMP dynamics independently of its kinase activity. A. Left panel: Mean change in intracellular cAMP concentration ([cAMP]i) in primary VSMCs expressing the TEpacVV biosensor from WT (top), PI3Kγ KO (center), and PI3Kγ-KD (bottom) mice. The black line represents the mean F480 / F535 emission ratio over time in response to 2.5 μM forskolin (fsk) stimulation and fsk (2.5 μM) + IBMX (200 μM) treatment. B. Histogram showing the mean forskolin response. Results are expressed as the percentage of the maximum ratio change (Rmax(IBMX response)) determined by the final application of forskolin (2.5 μM) + IBMX (200 μM). Data shown are mean ± SEM (n=5 mice). Data are expressed as mean ± SEM and compared using the Kruskal-Wallis test. Homogeneous time-resolved fluorescence (HTRF) levels of intracellular cAMP derived from WT, PI3Kγ KO, or PI3Kγ KD primary VSMCs after 30 minutes of treatment with C. vehicle or 0.5 μM forskolin. [Figure 3]In VSMCs, PI3Kγ controls cAMP levels via PDE4. A. Mean change in [cAMP]i in primary VSMCs expressing the TEpacVV biosensor from WT (top), PI3Kγ KO (middle), and PI3Kγ-KD (bottom) mice. Traces show the mean F480 / F535 radioactivity ratio over time in response to 2.5 μM forskolin, cilostamide (1 μM), rolipram (1 μM), and forskolin (2.5 μM) + IBMX (200 μM) treatments. B. Histograms showing the mean responses to rolipram and cilostamide. Results are expressed as the percentage of the maximum ratio change (Rmax(IBMX response)) determined by the final application of forskolin (2.5 μM) + IBMX (200 μM). Data are presented as mean ± SEM (n>22, 4 different mice) and compared using the Kruskal-Wallis test. C. Phosphodiesterase activity detected in PDE4D immunoprecipitation from primary VSMC. The data shown are mean ± SEM and compared using the Bonferroni post-hoc test after one-way ANOVA (n=3). D. Representative Western blot of PDE4D immunoprecipitation. [Figure 4] The permeable N-terminal peptide of PI3Kγ selectively inhibits the docking activity of PI3Kγ. A. Schematic diagram of a cell-permeable PI3Kγ competitive peptide. The 126-150 region of PI3Kγ was fused to the cell-permeable peptide, penetratin 1 (P1). Control peptides were generated by inserting two point mutations (K>A and R>A at positions 1 and 5 of the PI3Kγ sequence, respectively). B. Raw macrophages were pre-treated for 1 hour with vehicle, PI3Kγ peptide (10 μM), or PI3Kγ kinase inhibitor AS605240 (1 μM), and then treated with C5a (50 μM) for 5 minutes. Representative Western blot images (left) and quantification of the ratio (right) of phospho-AKT (Ser-473) and total AKT are shown. GAPDH was used as a loading control. n=3. C. Incorporation of FITC-labeled PI3Kγ peptide by primary VSMCs at 1, 5, 24, and 30 hours after incubation. D. Quantification of FITC-related fluorescence intensity within the VSMC region. [Figure 5] The permeable N-terminal peptide of PI3Kγ is the growth rate expressed as the doubling rate of primary VSMCs derived from WT (A), PI3Kγ KD (B), or PI3Kγ KO (C) aorta, measured by BrdU incorporation and compared to control. Cultures were incubated for 24 hours with 25 ng / ml PDGF with or without the addition of 25 μM forskolin in the presence of 25 μM control (white bar) or blocking peptide (black bar). BrdU was added during the last 18 hours (n = 8 cultures for each genotype). Data are presented as mean ± SEM and were compared using one-way ANOVA test. D. Schematic model of the involvement of PI3Kγ in VSMC growth. VSMC growth is induced by PDGF stimulation. The previous results of the inventors shown in gray indicate that the autocrine secretion of MCP1 mobilizes and activates PI3Kγ via GPCR signaling and controls cell migration after PDGF stimulation

[13] . This mobilization of PI3Kγ can also sequester phosphodiesterase (PDE), thereby counteracting cAMP production by adenylate cyclase (AC) induced by other signals. By these mechanisms, PI3Kγ acts as an amplification factor for VSMC synthetic migration. [Figure 6] Amino acid sequence of a peptide having the ability to permeate cell membranes.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] In the following description, numerous specific details are provided to provide a thorough understanding of the embodiments. Embodiments may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0013] Throughout this specification, the reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0014] The headings provided in this specification are for convenience only and are not to be construed as limiting the scope or meaning of the embodiments.

[0015] In one embodiment, the present invention relates to a fusion peptide for use in the treatment, prevention, and / or delay of onset of fibroproliferative vascular diseases, wherein the fusion peptide comprises (a) an amino acid sequence defined in SEQ ID NO: 1 or a related homolog having at least 85%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 1 and having the ability to inhibit the kinase-independent function of PI3Kγ, and (b) a peptide having the ability to penetrate cells and comprises.

[0016] In a further embodiment, the peptide having the ability to penetrate cells is selected from the sequences defined in SEQ ID NOs: 2- to 12, preferably SEQ ID NOs: 2- to 5, more preferably SEQ ID NO: 2.

[0017] In one embodiment, the fusion peptide can comprise a linker, preferably an amino acid linker, thereby enabling the linkage of the amino acid sequence defined in SEQ ID NO: 1 or a homolog thereof with the peptide having the ability to penetrate cells. However, the presence of a linker is not essential.

[0018] In yet a further embodiment, the peptide having the ability to penetrate cells is linked to the C-terminus or N-terminus of SEQ ID NO: 1 or a homolog thereof.

[0019] In further embodiments, the fusion peptide has the amino acid sequence described in SEQ ID NO: 14.

[0020] In further embodiments, the fusion peptides defined above are suitable for the treatment / prevention / delay of onset of fibroproliferative vascular diseases selected from restenosis, hypertension, pulmonary hypertension, preferably pulmonary arterial hypertension, atherosclerosis, and atherosclerosis (resulting in intimal thickening).

[0021] In one different embodiment, the present invention relates to a pharmaceutical composition for use in the treatment, prevention, and / or delay of the onset of fibroproliferative vascular disease, comprising a fusion peptide and a pharmaceutically acceptable vehicle, wherein the fusion peptide comprises (a) an amino acid sequence defined in SEQ ID NO: 1 or an associated homolog of SEQ ID NO: 1 having at least 85%, preferably at least 90%, more preferably at least 95% identity with SEQ ID NO: 1 and having the ability to inhibit the kinase-independent function of PI3Kγ, and (b) a peptide having the ability to permeate cells.

[0022] In one embodiment, the fusion peptide contained in the pharmaceutical composition comprises a peptide having the ability to penetrate cells, selected from the sequences defined in SEQ ID NOs: 2 to 12, preferably SEQ ID NOs: 2 to 5, more preferably SEQ ID NO: 2.

[0023] In one embodiment, the fusion peptide contained in the pharmaceutical composition comprises a peptide having the ability to penetrate cells, and such peptide is ligated to the C-terminus or N-terminus of SEQ ID NO: 1 or its homolog.

[0024] In one embodiment, the fusion peptide contained in the pharmaceutical composition may include a linker, preferably an amino acid linker, which enables linking of the amino acid sequence defined in SEQ ID NO: 1 or its homolog with a peptide having the ability to penetrate cells. However, the presence of a linker is not essential.

[0025] In a further embodiment, the pharmaceutical composition comprises a fusion peptide having the amino acid sequence described in SEQ ID NO: 14.

[0026] In one embodiment, the pharmaceutical composition is suitable for the treatment / prevention / delay of onset of fibroproliferative vascular diseases selected from restenosis, hypertension, pulmonary hypertension, preferably pulmonary arterial hypertension, atherosclerosis, and atherosclerosis (resulting in intimal thickening).

[0027] The present invention also discloses a method for treating / preventing / delaying the onset of fibroproliferative vascular disease, the method comprising administering to a patient in need at least one fusion peptide in an amount sufficient to perform the treatment, wherein the fusion peptide is a) an amino acid sequence defined in SEQ ID NO: 1 or an associated homolog having at least 85% similarity to SEQ ID NO: 1 and possessing the ability of SEQ ID NO: 1 to inhibit the kinase-independent function of PI3Kγ, and b) Peptides that have the ability to permeate cells Includes.

[0028] In various diseases such as atherosclerosis

[14] and pulmonary hypertension[15, 16], intimal vascular smooth muscle cells (VSMCs) proliferate abnormally, eventually leading to intimal hyperplasia (IH) and resulting in vascular occlusion. Angioplasty and metal stent implantation widen vessels narrowed by such VSMC overgrowth, but these procedures fail if VSMCs continue to proliferate and cause restenosis. Similarly, venous bypass grafting often fails due to VSMC proliferation into the new occluded intimal layer. Therefore, pharmacological reduction of VSMC proliferation is crucial in the treatment of vascular occlusion caused by IH.

[0029] Numerous studies suggest that intracellular increases of the signaling molecule cAMP play a crucial role in both maintaining vasoquiescence of vasoconstrictors (VSMCs) in healthy vessels and reducing restenosis in pathological compartments [6]. Small molecule inhibitors of phosphodiesterase (PDE), a cAMP hydrolase, have antiproliferative effects on VSMCs, and drugs such as cilostazol may have potential clinical therapeutic uses for the prevention of restenosis after arterial intervention

[17] . Limiting systemic exposure to such drugs has been difficult and ultimately led to unwanted side effects such as cardiac arrhythmias, thus hindering the large-scale adoption of such interventions. Furthermore, PDE is a large family of isozymes classified into 11 groups [6], and the lack of isoform-selective PDE inhibitors, particularly within the same family, has further limited the application of safe and effective cAMP elevation in the treatment of IH [6].

[0030] These results demonstrate that a cell-penetrating peptide containing residues 126-150 (SEQ ID NO: 1) of the N-terminal domain of the protein PI3Kγ can selectively increase cAMP in VSMCs by disrupting the function of a specific PDE isoform, PDE4D. This peptide (named KIT2014;

[12] ) disrupts the interaction between PI3Kγ and PKA, delocalizing PKA and reducing its ability to activate a PDE isoform specific to a selected cell type

[18] . The amino acid sequence of PI3Kγ found in KIT2014 has been previously shown to reduce arrhythmia-causing PDE3 activity in cardiomyocytes in vitro

[19] . Furthermore, this peptide has been shown to disrupt the PI3Kγ / PKA complex in airway smooth muscle, resulting in PDE4B / D inhibition and bronchorelaxation

[12] . VSMCs are similar to airway smooth muscle, but cannot be superimposed from a gene expression and cell biology standpoint [20, 21], thus complicating the prediction of peptide efficacy in these cells. Our results show that when conjugated with a cell-permeable peptide such as penetratin 1, this peptide sequence efficiently enters VSMCs and increases their cAMP levels.

[0031] It was impossible to predict from prior art whether KIT2014 could induce cAMP-dependent proliferation inhibition in VSMCs. Consensus on cAMP action demonstrates that intracellular signaling is complexly compartmentalized due to the existence of distinct pools of cAMP with separate functions within the same cell

[22] . The inventors' previous findings demonstrated that KIT2014 modulates the cAMP pools that control cardiac contraction and bronchorelaxation, respectively, in cardiomyocytes and airway smooth muscle. The data reported here confirm the existence of distinct functional compartments of cAMP signaling in smooth muscle

[23] , as well as the ability of KIT2014 to act on selected intracellular cAMP pools [12, 19]. These results demonstrate that KIT2014 specifically blunts PDE4D function, eliminating VSMC proliferation in response to PDGF and forskolin, and consequently demonstrates that the cAMP elevation induced by KIT2014 is sufficient to reduce proliferation and IH in relation to VSMCs.

[0032] Previous studies have shown that blocking PI3Kγ activity in VSMCs could demonstrate a novel therapeutic approach to treat IH [24, 25]. However, PI3Kγ is a bifunctional protein that possesses not only the ability to interact with PKA to regulate cAMP levels (scaffolding activity) but also the ability to produce lipid second messenger molecules through its catalytic activity. Unlike what is reported here, previous studies on PI3Kγ in VSMCs have focused on the catalytic role of the protein rather than its ability to interact with PKA to regulate cAMP levels. Yu et al.

[25] investigated the role of PI3Kγ catalytic activity in IH in arterial transplants and in phenotypic alterations of VSMCs induced by TNFα. This study teaches that inhibition of PI3Kγ catalytic activity with the selective inhibitor AS605240, as well as shRNA-mediated knockdown of PI3Kγ, yield the same protective effect, reducing IH overall in an aortic restenosis model

[25] . Both pharmacological and genetic inhibition of PI3Kγ resulted in a decrease in Akt phosphorylation, a well-established marker of PI3K catalytic activity. As an enzyme, PI3Kγ produces the second messenger molecule PIP3, which is essentially required to mediate Akt phosphorylation. From the finding that knockdown of the PI3Kγ gene yielded the same results as treatment with selective inhibitors of PI3Kγ kinase activity, Yu et al. concluded that PI3Kγ's ability to induce Akt phosphorylation via its kinase activity and its ability to produce PIP3 are crucial factors in IH. PI3Kγ also possesses a scaffolding function indirectly related to the regulation of another secondary messenger molecule, cAMP, independently of its kinase activity, but this is not mentioned by Yu et al.

[25] , and only the effects observed through the regulation of catalytic activity are explained.

[0033] Considering that PI3Kγ expression occurs not only within VSMCs but also in leukocytes, which can indirectly regulate VSMC responses and IH, the specific involvement of each of these two cell types was investigated in a model of PI3Kγ-dependent IH

[24] . In this secondary study, Yu et al. first confirmed that the kinase activity of PI3Kγ is involved in IH in response to vascular injury. In their model, both pharmacological inhibition and genetic knockdown of PI3Kγ were confirmed to reduce IH. They reported that a decrease in the catalytic activity of PI3Kγ blocks the phosphorylation and activation of Akt. Consistent with this effect, they also demonstrated that Akt-mediated phosphorylation of CREB and CREB-dependent transcriptional activation of YAP are determined by the catalytic activity of PI3Kγ. Furthermore, to address the question of which cell types are involved in this process, they showed that specific downregulation of PI3Kγ in either leukocytes or vascular tissue occurs concurrently with protection against IH progression in response to vascular injury

[24] . All of these studies have focused on the catalytic activity of PI3Kγ and have not investigated whether the scaffolding function of this protein and cAMP levels are involved.

[0034] In contrast, the data provided herein clearly demonstrate that the kinase-independent function of PI3Kγ influences VSMC proliferation through a direct cell-autonomous mechanism. This data definitively distinguishes between the kinase-dependent and kinase-independent roles of PI3Kγ in vivo, as demonstrated by studies of genetically engineered mice (PI3Kγ-KD mice) expressing a kinase-dead version of the enzyme that retains its ability to interact with PKA

[19] . Results from this study using PI3Kγ-KD mice clearly demonstrated that loss of catalytic activity in the hematopoietic compartment resulted in a small decrease in IH. Conversely, loss of PI3Kγ catalytic activity in the non-hematopoietic compartment failed to inhibit IH. Consistent with the role of protein-protein interactions between PI3Kγ and PKA, complete loss of PI3Kγ in KO mice resulted in a large decrease in IH in vivo. Further evidence of the involvement of PI3Kγ's docking function in regulating VSMC proliferation is found in vitro in a peptide (KIT2014) that specifically targets the scaffold but not PI3Kγ catalytic activity. Consistent with this view, the ability of KIT2014 to block VSMC proliferation is confirmed to act independently of PI3Kγ catalytic activity, demonstrating its ability to independently target the two roles of the enzyme.

[0035] In short, these results are consistent with the unprecedented role of KIT2014 in disrupting the PI3Kγ / PKA complex, which promotes PDE4D-dependent cAMP inhibition necessary for maintaining VSMC proliferation. Based on this, our findings point to non-obvious uses of KIT2014 in the treatment of IH occurring in conditions such as restenosis, atherosclerosis, and pulmonary hypertension. KIT2014 can be used as a component of a topical treatment accompanying a medical stent or angioplasty for the treatment of atherosclerosis and restenosis, or as an inhalant for the treatment of pulmonary hypertension. [Examples]

[0036] [result] The inventors' previous results identified the catalytic role of PI3Kγ in the immune processes of IH progression and arterial healing

[26] . This was demonstrated by using knock-in mice (PI3Kγ-KD) expressing catalytically inactive PI3Kγ in mechanically damaged arteries. PI3Kγ KD mice showed reduced arterial occlusion and monocyte and T cell accumulation around vascular lesion sites. In this study, the inventors aimed to further investigate the involvement of PI3Kγ in the vascular compartment and to evaluate the possible role of its non-catalytic docking function in IH. Bone marrow

[27] chimeras were produced using PI3Kγ-KO and PI3Kγ-KD mice as recipients to distinguish between kinase-dependent and kinase-independent functions of PI3Kγ in the vascular compartment. The inventors introduced WT-derived BM into PI3Kγ-KO (WT>KO) or PI3Kγ-KD (WT>KD) animals, and vice versa (KO>WT and KD>WT). After grafting, the femoral artery was injured, and the lesions were analyzed 28 days later. In BM WT>KO and KO>KO chimeras, the intima / media ratio was reduced by 40% compared to BM WT>WT chimeras (Figure 1A), but no difference was observed in BM WT>KD chimeras, as previously observed

[26] . These results suggest that the presence of PI3Kγ (not its catalytic activity) is required to induce a fibroproliferative response to injury in the vascular compartment.

[0037] Consistent with the inventors' previous results

[26] , BM KO>WT, BM KD>WT, or BM KD>KD chimeras showed a significant reduction in the intima / media ratio, confirming the role of PI3Kγ catalytic activity in the immune compartment (Figure 1A). Histological analysis of injured arteries by Masson Trichrome staining showed a reduction in neointimal area in BM WT>KO and BM KO>WT chimeras compared to controls (Figure 1A). This staining clearly indicated that the reduction in neointimal area in BM WT>KO was mainly due to less VSMC layer expansion (Figure 1A). Taken together, these results suggest that post-injury VSMC proliferation is independent of the catalytic function of PI3Kγ, and rather involves its docking function.

[0038] Considering the importance of cAMP in inhibiting VSMC proliferation in vitro and in vivo [3-5], we hypothesized that the kinase-independent function of PI3Kγ may also be involved in VSMC proliferation via cAMP regulation. To test this hypothesis, we evaluated the proliferation of primary VSMCs from different genotypes (WT, PI3Kγ-KO, or PI3Kγ-KD) by stimulation with platelet-derived growth factor (PDGF), a potent mitogenic agent for VSMCs. Forskolin (FSK), an adenylate cyclase activator, was supplemented into the culture medium to increase cAMP levels and counteract PDGF-induced VSMC proliferation (Figure 1C). Our results showed that 25 μM FSK reduced VSMC proliferation, and the FSK effect was significantly enhanced in the absence of PI3Kγ (PI3Kγ-KO) but not in PI3Kγ-KD VSMCs, confirming the role of PI3Kγ's AKAP function in VSMCs (Figures 1D and E). These results demonstrate that PI3Kγ controls VSMC proliferation by regulating the intracellular concentration of cAMP, independently of its enzymatic activity.

[0039] The involvement of PI3Kγ in the regulation of intracellular cAMP concentration in VSMCs is based on FRET. T Epac VVThe dynamics of cAMP signaling in primary VSMCs of WT, PI3Kγ-KD, or PI3Kγ-KO were confirmed by using a biosensor

[28] . The relative change in cAMP levels in response to 2.5 μM forskolin was monitored over time by calculating the ratio of donor fluorescence (F480 nm) to acceptor fluorescence (F535 nm). The cAMP level was determined as a percentage of the maximum ratio change (Rmax). Rmax was determined at the end of the experiment by adding 200 μM 3-isobutyl-1-methylxanthine (IBMX) (a pan-PDE inhibitor) in the presence of 2.5 μM forskolin. As shown in Figure 2, cAMP production in response to 2.5 μM forskolin increased 2.2-fold in PI3Kγ-KO cells compared to WT primary VSMCs (24.09% vs. 52.57% IBMX response, p=0.03), while the forskolin response in PI3Kγ-KD cells remained similar to that of WT cells (Figures 2A-C). Basal cAMP levels remained stable and unchanged in all three genotypes (Figures 2A and B). Furthermore, the change in maximum ratio was also identical in VSMCs derived from all genotypes (Figure 2A). Taken together, these results indicate that the cAMP response to forskolin is higher in PI3Kγ-KO VSMCs than in WT and PI3Kγ-KD cells, demonstrating the role of PI3Kγ in limiting cAMP levels.

[0040] Intracellular levels of cAMP depend on their production and degradation (by adenylyl cyclase (AC) and PDE, respectively). PI3Kγ has already been shown to bind to PDE in cardiomyocytes [11, 19], and of these, the inventors focused on PDE3 and PDE4 because they are involved in IH [8, 10, 29]. They sequentially treated forskolin-stimulated VSMCs with cilostamide and rolipram (inhibiting PDE3 and PDE4, respectively) and quantified cAMP levels according to Rmax. These experiments allowed them to assess the relative proportion of the cAMP pool regulated by each PDE in different genotypes. The results showed that cAMP levels were primarily regulated by PDE4 in WT and PI3Kγ-KD cells (67% and 73%, respectively, compared to the IBMX response). The involvement of PDE3 in cAMP level regulation in these cells was limited to 25% and 17%, respectively, compared to the IBMX response (Figure 3A, B, and C). Therefore, in PI3Kγ-KO cells, the main PDE isoform that degrades cAMP is PDE3 (49% of IBMX responses for PDE3, and 43% for PDE4, Figure 2C). To confirm the involvement of the PDE4 enzyme in PI3Kγ-downstream cAMP regulation, the inventors performed immunoprecipitation and measured PDE4 activity. They focused on PDE4D, the main isoform expressed in VSMCs. Consistent with the inventors' cAMP measurements in living cells, PDE4D activity was significantly reduced in PI3Kγ-KO cells compared to WT and PI3Kγ-KD cells (Figures 3D and E).

[0041] PI3Kγ has been shown to regulate cAMP levels and signaling in cardiomyocytes via its AKAP function

[19] . Interestingly, the interaction between PI3Kγ and the PKA / PDE complex is mapped to amino acids 126–150 of the N-terminal region of p110γ

[19] . To further investigate the functional relationship between PI3Kγ, PKA / PDE-mediated cAMP regulation and VSMC proliferation, we disrupted the PI3Kγ / PKA interaction by treating cells with a cell permeability competitive peptide corresponding to the PKA binding region on p110γ and examined their proliferation in response to PDGF and forskolin

[19] . We designed control peptides with two point mutations that do not interact with PKA (K>A and R>A at positions 1 and 5 of the PI3Kγ sequence, respectively)

[19] (Figure 4A). First, we evaluated the ability of peptides to selectively interfere with the PI3Kγ scaffold without interfering with catalytic function. They found that C5a-mediated Akt phosphorylation in macrophages (an event dependent on the catalytic activity of PI3Kγ

[30] ) remained unchanged in cells treated with the PI3Kγ peptide, but was significantly reduced in cells exposed to the standard PI3Kγ kinase inhibitor AS-6052540 (Figure 4B). Next, they determined the time course of peptide permeability in VSMCs using a FITC-tagged version of the peptide. The peptide was detectable in cells after 5 hours and even after 30 hours of incubation (Figures 4C-D). Therefore, the inventors used the PI3Kγ peptide to block the PI3Kγ-PKA interaction in cell proliferation experiments, as shown in Figure 1D. Incubation with the blocking peptide inhibited cell proliferation in WT and PI3Kγ-KD cells (Figures 5A and B), but no difference was observed in PI3Kγ-KO cells (Figure 5C). These data clearly demonstrate that PI3Kγ controls VSMC proliferation through its AKAP function.

[0042] In summary, these results suggest that PI3Kγ acts as a catalyst for VSMC proliferation by reducing cAMP levels, primarily through the direct regulation of the PDE4 enzyme (Figure 5D).

[0043] [method] Animals: PI3Kγ-deficient (PI3Kγ-KO) mice and mice expressing a catalytically inactive form of PI3Kγ (PI3Kγ kinase dead mice; PI3Kγ-KD) were derived from the C57BL / 6J background and have been previously described [31-33]. WT, PI3Kγ-KD, and PI3Kγ-KO mice were maintained at the Rangueil Animal Facility (UMS 06; Anexplo platform) under SPF conditions. All animal procedures were carried out in accordance with institutional guidelines for animal experiments and under a French Ministry of Agriculture license.

[0044] Femoral artery wire injury in mice: WT, PI3Kγ-KD, and PI3Kγ-KO male mice (8-10 weeks old) were studied for IH research using a proven model of femoral artery wire injury

[27] . Briefly, general anesthesia was achieved using 2% isoflurane. The femoral artery was then isolated and incised under a surgical microscope (Carl Zeiss). A 0.35 mm diameter angioplasty guidewire with a 0.25 mm tip was advanced into the artery three times to the level of the arterio-aortic bifurcation and then withdrawn. After wire removal, the arterial incision site was ligated. Mice were sacrificed 28 days after injury for histological and immunohistochemical analysis.

[0045] Tissue processing and morphometry: IH quantification was performed by paraffin embedding technique. At the time of euthanasia, mice were perfused with PBS followed by 4% paraformaldehyde. Blood vessels were harvested and fixed in 4% formalin (pH = 8) for 24 hours. They were embedded in paraffin and prepared as slides (4-μm thick sections). To quantify the IH ratio, sections were stained with Masson Trichrome (Bio Optica) and then the lengths (μm) around the lumen, IEL (internal elastic lamina), and EEL (external elastic lamina) were measured in 4 sections per blood vessel using LAS software (Leica) at 0.5, 2.0, 3.5, and 5 mm from the ligation site. Arteries that had undergone occlusive thrombotic events were excluded from the quantification group. The area (μm 2 ) defined by EEL was calculated assuming an in vivo circular shape: (A EEL = EEL circumference 2 ÷4π). The area (μm 2 ) defined by IEL was calculated using (A IEL = IEL circumference 2 ÷4π). The lumen area (μm 2 ) was calculated using (A LUM = lumen circumference 2 ÷4π). The neointimal area (μm 2 ) was calculated using (A NEO = A IEL - A LUM ). The medial area (μm 2 ) was calculated using (A MED = A EEL - A IEL ). The intima / media ratio was calculated using (neointima / media = A NEO ÷A[[ID=3S]] MED ). <00002CI> Bone marrow (BM) transplantation: BM was obtained from 8-week-old WT, PI3Kγ-KD, and PI3Kγ-KO mice. BM cells were flushed from femurs and tibias, then washed, filtered, counted, and 107 Unfractionated cells were resuspended in sterile PBS for post-orbital injection. Four weeks later, the femoral artery was damaged, and the mice were euthanized 28 days after the surgical procedure. Successful engraftment was confirmed by PCR.

[0047] Isolation of SMCs from Muhlin's aorta and cultures: Mouse VSMCs were isolated from WT mice according to the modified protocol described in

[34] . Briefly, the aortas from 4WT mice were dissected from their origin to the iliac bifurcation and flushed with PBS. The outer membrane was removed from the aorta, the smooth tube was cut into 1-2 mm pieces, and then digested in 0.3% collagenase solution. Collagenase digestion was stopped by adding DMEM (D0822, Sigma Aldrich) / 10% FBS (FBS, Thermo Fisher Scientific). The aortic pieces were then washed twice and seeded in culture dishes. Primary confluent cultures were trypsinized at 37°C (0.1% trypsin; Thermo Fisher Scientific), and the cells were incubated in DMEM / 10% FBS in 5% CO2 at 37°C and cultured up to passage 4.

[0048] Measurement of mouse SMC proliferation: Primary mouse SMCs were subcultured in complete medium on 48-well cell culture plates. Cells were treated with PDGF (520-BB-050, R&D Systems) and forskolin (F3917, Sigma Aldrich) for 24 hours, or not. For blocking peptide experiments, cells were incubated for 24 hours with 25 μM control (RQIKIWFQNRRMKWKKGAATHASPGQIHLVQRHPPSEESQAF-SEQ ID NO: 13) or blocking peptide (RQIKIWFQNRRMKWKKGKATHRSPGQIHLVQRHPPSEESQAF-SEQ ID NO: 14). Peptides were synthesized by GenScript (GenScript, Piscataway, NJ). BrdU (00-0103, Invitrogen) uptake assays were performed using a classical immunofluorescence procedure with anti-BrdU antibody (11-5071, eBioscience) and DAPI. The proliferation rate was measured as the ratio of BrdU-positive nuclei to the total nucleus and expressed as an increase factor.

[0049] Measurement of cAMP dynamics: Primary SMC was sown on coverslips and as described elsewhere

[35] , T Epac VV The coverslips were infected with type 5 adenovirus (approximately 100 particles / SMC) encoding [the specified virus]. The coverslips were continuously perfused with BBS buffer saturated with 95% O2-5% CO2 (125 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 25 mM glucose) (2 mL·min). -1The cells were placed in a microscope chamber. Ratiometric analysis was performed as follows: fluorescence was excited using a 435 nm LED source, and fluorescence emission was monitored using a dichroic mirror (T450LPXR) and alternating emission filters of donor (HQ480 / 40) and acceptor (D535 / 40). Image pairs were recorded at 20-second intervals using an Orca-ER CCD camera (Hamamatsu Photonics, Japan). The change in [cAMP]i is expressed as the ratio of donor fluorescence (F480) to acceptor fluorescence (F535). The same constant was multiplied by the ratio for all experiments so that the baseline ratio under ground conditions was 1. The maximum ratio change (Rmax) was obtained by stimulating the cells with 2.5 μM forskolin and 200 μM IBMX (I5879, Sigma Aldrich). The filters and mirrors were obtained from AHF Analysttechnik AG, Tubingen, Germany.

[0050] Measurement of PDE activity: Cells were scraped into 120 mmol / L NaCl, 50 mmol / L Tris-HCl (pH 8.0), and 1% Triton X-100 supplemented with protease and phosphatase inhibitors, and centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatant was immunoprecipitated and then analyzed for PDE activity or / or Western blotting. For the immunoprecipitation assay, 200 μg of pre-cleared extract was incubated with 20 μl of a 1:1 slurry of protein A- or protein G-Sepharose (Amersham Biosciences, Buckinghamshire, UK) and 1 μg of anti-PDE4D antibody (Abcam#ab171750) for 2 hours at 4°C. The immunocomplex was then broadly washed with lysis buffer and the PDE activity assay was performed. PDE activity in immunoprecipitation was measured according to a slightly modified two-step method of Thompson and Appleman. Briefly, the immunoprecipitation was subjected to 40 mmol / L Tris-HCl (pH 8.0), 1 mmol / L MgCl2, 1.4 mmol / L 2-mercaptoethanol, 1 μmol / L cAMP (Sigma-Aldrich, Saint Louis, MO), and 0.1 μCi [ 3 The assay was performed at 33°C for 45 minutes in a total volume of 200 μl of reaction mixture containing [H]cAMP (Amersham Bioscience, Buckinghamshire, UK). To stop the reaction, the sample was boiled at 95°C for 3 minutes. The PDE reaction product 5'-AMP was then hydrolyzed by incubating the assay mixture with 50 μg of Crotalus Atrox snake venom at 37°C for 15 minutes (Sigma-Aldrich, Saint Louis, MO). The resulting adenosine was separated by anion exchange chromatography using 400 μl of a 30% (w / v) suspension of Dowex AG1-X8 resin (Bio-Rad, Segrate, Milano, Italy). The amount of radiolabeled adenosine in the supernatant was quantified by scintillation (Ultima Gold scintillation solution by Perkin Elmer, Waltham, MA).

[0051] C5a-mediated Akt phosphorylation in macrophages: After 3 hours of starvation in serum-free medium, RAW264 macrophages were pre-treated for 30 minutes with PI3Kγ competitive peptide (25 μM), PI3Kγ inhibitor AS-605240 (1 μM; MedChemExpress), or DMSO, and then stimulated with 50 nM C5a (Sigma Aldrich) for 5 minutes. Akt phosphorylation at Ser-473 was detected by Western blotting using a Ser473 Akt-specific antibody (Cell Signaling Technology).

[0052] [References] JPEG0007886826000001.jpg237166JPEG0007886826000002.jpg235166JPEG0007886826000003.jpg238166

Claims

1. A pharmaceutical composition for the treatment, prevention, and / or delay of the onset of fibroproliferative vascular disease, It comprises a fusion peptide and a pharmaceutically acceptable vehicle. The fusion peptide substantially comprises (a) the amino acid sequence defined in SEQ ID NO: 1, or an associated homolog having at least 95% identity with the amino acid sequence defined in SEQ ID NO: 1 and having the ability to inhibit the kinase-independent function of PI3Kγ, and (b) a peptide having the ability to permeate cells. Pharmaceutical composition.

2. A pharmaceutical composition according to claim 1, The peptide having the ability to penetrate the aforementioned cells is selected from the sequences defined in Sequence IDs 2 to 12. Pharmaceutical composition.

3. A pharmaceutical composition according to claim 1 or 2, The peptide having the ability to penetrate the aforementioned cells is linked to the C-terminus or N-terminus of the amino acid sequence defined in Sequence ID No. 1 or its homolog. Pharmaceutical composition.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the fusion peptide has the amino acid sequence described in SEQ ID NO:

14.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the fibroproliferative vascular disease is selected from restenosis, hypertension, pulmonary hypertension, pulmonary arterial hypertension, atherosclerosis, and atherosclerosis.

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