Patent Description:
Cardiovascular diseases represent a significant part of morbidity and mortality worldwide. These pathologies often originate from vascular dysfunction and occlusion, occurring even after interventional strategies used in the treatment of atherosclerosis. For instance, intimal hyperplasia (IH) ensues from atherosclerotic plaque development but also after transluminal angioplasty and stent positioning, a standard treatment of atherosclerosis. IH consists in a complex multifactorial response of the vessel wall resulting in the formation of a multicellular layer within the arterial lumen. It involves inflammatory and fibroproliferative phenomena due to endothelial injury and subsequent vascular smooth muscle cell (VSMC) dedifferentiation, leading to their over-proliferation and migration. Together, these processes end up with intimal thickening and a reduction in blood flow, which can have dramatic consequences. Thus, understanding mechanisms of VSMC proliferation is critical for the development of new therapeutic strategies to prevent IH [<NUM>, <NUM>].

Among the signaling molecules involved in VSMC proliferation, the second messenger <NUM>'-<NUM>' cyclic adenosine monophosphate (cAMP) plays a major role. In VSMC, elevated cAMP levels inhibit mitogen-stimulated proliferation both in vitro and in vivo [<NUM>-<NUM>]. Consistently, cAMP has been found to have a positive role in reducing intimal thickening in several models of vascular injury. Moreover, cAMP signaling is affected in a large variety of vascular pathologies including atherosclerosis or restenosis after angioplasty. cAMP signals mainly depend on its localized production by adenylyl cyclases (AC) and its degradation by cyclic nucleotide phosphodiesterases (PDE) [<NUM>]. In VSMC, PDE3A, PDE4 and PDE1C have been implicated in the regulation of VSMC proliferation and IH [<NUM>, <NUM>, <NUM>]. Interestingly, it has been shown that cAMP production is linked to an increase of PDE4 activity and upregulated expression of the PDE4D isoform in both contractile and synthetic phenotypes [<NUM>]. Consistently, numerous studies have been performed using PDE inhibitors in various arterial disease models [<NUM>].

In this context, phosphoinositide <NUM>-kinase gamma (PI3Kγ), a lipid kinase involved in immune regulation of cardiovascular diseases, has been described to regulate cAMP levels in cardiomyocytes through a kinase-independent mechanism. While, on the one hand, PI3Kγ can generate <NUM>-phosphoinositides lipid second messengers as a kinase, on the other hand, it can act as a "A-kinase anchoring protein" (AKAP) associating, within the same macromolecular complex, with cAMP-dependent kinase, also known as protein kinase A (PKA), and phosphodiesterase <NUM> and <NUM> (PDE3 and PDE4), PKA-controlled enzymes responsible for cAMP degradation. In line with this view, the catalytic subunit of the PI3Kγ holoenzyme (p110γ) interacts with PKA and participates in a negative feedback loop, leading to PDE3 and PDE4 activation and eventually to increased cAMP degradation.

In cardiac cells, PI3Kγ restrains cAMP elevation in response to beta-adrenergic stimulation, ultimately reducing cardiac contractility [<NUM>]. On the contrary, PI3Ky-mediated regulation of cAMP levels in airway smooth muscle cells promotes contraction, leading to bronchoconstriction. Accordingly, the inhibition of the kinase-independent function of PI3Kγ in airway smooth muscles with a cell-permeable competing peptide promotes bronchorelaxation [<NUM>].

The object of this disclosure is to provide a new strategy for the treatment/prevention/delay of onset of fibroproliferative vascular diseases.

According to the invention, the above object is achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure.

The present invention concerns a fusion peptide, and a pharmaceutical composition containing a fusion peptide for use in treating, preventing and/or delaying onset of a fibroproliferative vascular disease, wherein the fusion peptide comprises:.

Arterial remodeling found in hypertension and intimal hyperplasia involves inflammation and disrupted flow, both contributing to smooth muscle cells dedifferentiation and proliferation. In the present application, the inventors identified for the first time a kinase-independent key role of non-hematopoietic phosphoinositide <NUM>-kinase gamma (PI3Kγ) in the vascular wall during intimal hyperplasia using mouse models deleted for PI3Kγ or expressing a Kinase Dead version of the enzyme. Moreover, the inventors found that absence of PI3Kγ in VSMC leads to a modulation of cell proliferation linked to an increase in intracellular cAMP levels. Real time analysis of cAMP dynamics reveals that PI3Kγ modulates the degradation of cAMP independently of its kinase activity in primary VSMC through the regulation of phosphodiesterase (PDE) <NUM> enzymes. The present invention thus concerns the therapeutic application of the N-terminal competing peptide of PI3Kγ (having the amino acid sequence set forth in SEQ ID No.: <NUM>) or a corresponding homolog that is able to block primary VSMC proliferation. These data provide evidence for a kinase-independent role of PI3Kγ in arterial remodeling and reveal novel strategies targeting the docking function of PI3Kγ for the treatment/prevention/delay of onset of fibroproliferative vascular diseases.

The invention will now be described in detail, purely by way of an illustrative and non-limiting example and, with reference to the accompanying drawings, wherein:.

In the description that follows, numerous specific details are given to provide a thorough understanding of the embodiments.

In one embodiment, the present invention concerns a fusion peptide for use in treating, preventing and/or delaying onset of a fibroproliferative vascular disease, wherein the fusion peptide comprises:.

In a further embodiment, the peptide having the ability to penetrate a cell is selected from the sequences specified in SEQ ID No.: <NUM> to <NUM>, preferably SEQ ID No.: <NUM> to <NUM>, more preferably SEQ ID No.: <NUM>.

In one embodiment, the fusion peptide can comprise a linker, preferably an amino acid linker, that allows the linking of the amino acid sequence as defined in SEQ ID No.: <NUM> or its homolog with the peptide having the ability to penetrate a cell. The presence of the linker is however not mandatory.

In a still further embodiment, the peptide having the ability to penetrate a cell is linked to the C-terminal or the N-terminal of SEQ ID No.: <NUM> or its homologs.

In a further embodiment, the fusion peptide has an amino acid sequence as set forth in SEQ ID No.: <NUM>.

In a further embodiment, the fusion peptide as defined above is suitable for the treatment/prevention/delay of onset of a fibroproliferative vascular disease selected from restenosis, hypertension, pulmonary hypertension, preferably pulmonary arterial hypertension, atherosclerosis and plaque rupture caused by intimal thickening.

In a different embodiment, the present invention concerns a pharmaceutical composition for use in treating, preventing and/or delaying onset of a fibroproliferative vascular disease containing a fusion peptide, and a pharmaceutically acceptable vehicle, wherein the fusion peptide comprises (a) an amino acid sequence as defined in SEQ ID No.: <NUM> or a related homolog having at least <NUM>%, preferably at least <NUM>%, more preferably at least <NUM>% identity with SEQ ID No.: <NUM> and having the ability of the sequence SEQ ID No.: <NUM> to inhibit the kinase-independent function of PI3Kγ, and (b) a peptide having the ability to penetrate a cell.

In one embodiment, the fusion peptide contained in the pharmaceutical composition comprises a peptide having the ability to penetrate a cell selected from the sequences specified in SEQ ID No.: <NUM> to <NUM>, preferably SEQ ID No.: <NUM> to <NUM>, more preferably SEQ ID No.: <NUM>.

In a preferred embodiment, the fusion peptide contained in the pharmaceutical composition comprises a peptide having the ability to penetrate a cell, wherein such a peptide is linked to the C-terminal or the N-terminal of SEQ ID No.: <NUM> or its homologs.

In a preferred embodiment, the fusion peptide contained in the pharmaceutical composition can comprise a linker, preferably an amino acid linker, that allows the linking of the amino acid sequence as defined in SEQ ID No.: <NUM> or its homolog with the peptide having the ability to penetrate a cell. The presence of the linker is however not mandatory.

In a further embodiment, the pharmaceutical composition comprises a fusion peptide having an amino acid sequence as set forth in SEQ ID No.: <NUM>.

In a preferred embodiment, the pharmaceutical composition is suitable for the treatment/prevention/delay of onset of a fibroproliferative vascular disease selected from restenosis, hypertension, pulmonary hypertension, preferably pulmonary arterial hypertension, atherosclerosis and plaque rupture caused by intimal thickening.

Also disclosed but not claimed is a method for treating/preventing/delaying onset of a fibroproliferative vascular disease comprising administering to a patient in need thereof at least one fusion peptide in an amount sufficient to carry out said treatment, wherein the fusion peptide comprises:.

In various diseases like atherosclerosis [<NUM>] and pulmonary arterial hypertension [<NUM>, <NUM>], intimal vascular smooth muscle cells (VSMC) abnormally proliferate, eventually triggering intimal hyperplasia (IH) leading to vessel occlusion. Angioplasty and metal stent implantation open vessels narrowed by such VSMC overgrowth, but this treatment fails if VSMC continue to expand and trigger restenosis. Similarly, vein by-pass grafts are frequently unsuccessful owing to the growth of VSMC into a new occlusive intimal layer. Thus, pharmacological reduction of VSMC proliferation is of critical importance in the treatment of vascular occlusion due to IH.

A large body of literature suggests that intracellular increase of the signaling molecule cAMP plays an important role in both the maintenance of VSMC quiescence in healthy vessels and the reduction of restenosis in diseased vascular districts [<NUM>]. Small molecule inhibitors of phosphodiesterases (PDE), the cAMP hydrolyzing enzymes, have an anti-proliferative effect on VSMC, and drugs like cilostazol may have potential for clinical therapeutic use for the prevention of restenosis following arterial intervention [<NUM>]. The difficulty of limiting systemic exposure to such drugs, eventually leading to unwanted side effects, like cardiac arrythmia, prevented large-scale adoption of such interventions. In addition, PDEs are a large family of isoenzymes, classified in <NUM> groups [<NUM>], and the lack of isoform-selective PDE inhibitors, especially within members of the same family, further restrained the application of safe and effective cAMP elevation, in the treatment of IH [<NUM>].

The present results indicated that a cell permeable peptide containing residues between <NUM> and <NUM> of the N-terminal domain of the protein PI3Kγ (SEQ ID No.: <NUM>) can selectively increase cAMP in VSMC by disturbing the function of a specific PDE isoform, PDE4D. This peptide (named KIT2014; [<NUM>]) disrupts the interaction between PI3Kγ and PKA, delocalizing PKA and reducing its ability to activate selected cell-type specific PDE isoforms [<NUM>]. The amino acid sequence of PI3Kγ found in KIT2014 has been previously shown to decrease PDE3 activity causing arrythmia in vitro in cardiomyocytes [<NUM>]. Moreover, this peptide has been shown to disrupt the PI3Kγ/PKA complex in airway smooth muscles, leading to PDE4B/D inhibition and bronchorelaxation [<NUM>]. VSMC, though being similar to airway smooth muscles, are not superimposable in terms of gene expression and cell biology [<NUM>, <NUM>], thus complicating predictions of peptide efficacy in these cells. The present results demonstrate that, when conjugated with a cell-penetrating peptide like Penetratin <NUM>, this peptide sequence efficiently enters VSMC and increases their cAMP levels.

Whether KIT2014 was able to trigger cAMP-dependent proliferative arrest in VSMC was impossible to predict from prior art. Consensus on cAMP way of action evidences complex subcellular signaling compartmentalization due to the existence, in the same cell, of different pools of cAMP with distinct functions [<NUM>]. Inventors' previous finding demonstrated that in cardiomyocytes and in airway smooth muscles KIT2014 regulates pools of cAMP which control cardiac contraction and bronchorelaxation, respectively. Data reported here confirm the presence of distinct functional compartments of cAMP signaling in smooth muscles [<NUM>] as well as the ability of KIT2014 to act on selected subcellular cAMP pools [<NUM>, <NUM>]. The present results, indicating that KIT2014 specifically blunted PDE4D function and abolished VSMC proliferation in response to PDGF and forskolin, conclusively demonstrated that, in the context of VSMC, the cAMP elevation elicited by KIT2014 is sufficient to reduce proliferation and IH.

Previous studies indicate that blocking PI3Kγ activity in VSMC could represent a new therapeutic approach to treat IH [<NUM>, <NUM>]. However, PI3Kγ is a bifunctional protein endowed not only with the ability to interact with PKA and modulate cAMP levels (scaffold activity) but also to generate lipid second messenger molecules through its catalytic activity. Differently from what reported here, previous studies on PI3Kγ in VSMC focused on the catalytic role of the protein rather than its ability to interact with PKA and modulate cAMP levels. [<NUM>] explored the role of PI3Kγ catalytic activity in IH of arterial grafts and in TNF-α-induced phenotypic switch of VSMC. This study teaches that inhibition of PI3Kγ catalytic activity with the selective inhibitor AS605240 as well as shRNA-mediated knockdown of PI3Kγ result in the same protective effects that overall reduce IH in a model of aortic restenosis [<NUM>]. Both pharmacological and genetic inhibition of PI3Kγ led to reduced Akt phosphorylation, a well-established marker of PI3K catalytic activity. As an enzyme, PI3Kγ generates the second messenger molecule PIP3, that in turn is essentially required to mediate Akt phosphorylation. The finding that knockdown of the PI3Kγ gene provides the same result of the treatment with the selective inhibitor of the PI3Kγ kinase activity, let Yu et al. conclude that the key driver in IH is the ability of PI3Kγ to trigger Akt phosphorylation through its kinase activity and the ability to produce PIP3. Although PI3Kγ possesses a scaffold function unrelated to its kinase activity and indirectly linked to the regulation of the other secondary messenger molecule cAMP, this is not mentioned by Yu et al. [<NUM>] that explain the observed effects through the modulation of the catalytic activity only.

Given that the expression of PI3Kγ occurs not only in VSMC but also in leukocytes that can indirectly modulate VSMC responses and IH, the specific involvement of each of these two cell types was explored in models of PI3Kγ-dependent IH [<NUM>]. In this second study, Yu et al. first confirm that the kinase activity of PI3Kγ is involved in IH in response to vascular injury. In their model they corroborate that both pharmacological inhibition and genetic knockdown of PI3Kγ reduce IH. They report that reduction of the catalytic activity of PI3Kγ blocks phosphorylation and activation of Akt. In line with this effect, they also demonstrate that Akt-mediated phosphorylation of CREB as well as CREB-dependent transcriptional activation of YAP are determined by the catalytic activity of PI3Kγ. In addition, to address the question of the specific cell type involved in this process, they show that specific downregulation of PI3Kγ in either leukocytes or vascular tissue concur to the protection against IH development in response to vascular injury [<NUM>]. All these studies converge on the catalytic activity of PI3Kγ and fail to explore whether the scaffold function of this protein and cAMP levels are involved.

On the contrary, data presented herein clearly demonstrate that the kinase independent function of PI3Kγ affects proliferation of VSMC through a direct, cell-autonomous mechanism. The present data conclusively discriminate between a kinase-dependent and a kinase-independent role of PI3Kγ in vivo through the study of mice genetically engineered to express a kinase-dead version of the enzyme (PI3Kγ-KD mice) that retains the ability to interact with PKA [<NUM>]. The present results with PI3Kγ-KD mice clearly demonstrated that loss of the catalytic activity in the hematopoietic compartment led to a small reduction of IH. Conversely, loss of the PI3Kγ catalytic activity in the non-hematopoietic compartment was not able to block IH. In line with a role of the protein-protein interaction between PI3Kγ and PKA, complete loss of PI3Kγ in KO mice led to a strong in vivo reduction of IH. As a further prove of the involvement of the docking function of PI3Kγ in the control of VSMC proliferation, a peptide specifically targeting the scaffold but not the catalytic activity of PI3Kγ (KIT2014) inhibits VSMC proliferation in vitro. In line with this view, the ability of KIT2014 to block VSMC proliferation acts independently of PI3Kγ catalytic activity and confirms that the two roles of the enzyme can be independently targeted.

In summary, the present results are consistent with an unprecedented role for KIT2014 in disrupting the PI3Kγ/PKA complex promoting the PDE4D-dependent cAMP suppression required to maintain VSMC proliferation. On this basis, the inventors' findings point to a non-obvious use of KIT2014 in the treatment of IH, occurring in disease like restenosis, atherosclerosis and pulmonary arterial hypertension. KIT2014 can be used as a component of medicated stents or local treatments concomitant with angioplasty for the treatment of atherosclerosis and restenosis, or as an inhaled agent for the treatment of pulmonary arterial hypertension.

Inventors' previous results identified a role of the catalytic activity of PI3Kγ in immune processes of IH development and arterial healing [<NUM>]. This was demonstrated by using knock-in mice expressing a catalytically inactive PI3Kγ (PI3Kγ-KD) subjected to arterial mechanical injury. PI3Kγ KD mice showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. In the present study the inventors intended to further explore the implication of PI3Kγ in the vascular compartment, and to evaluate a possible role of its non-catalytical and docking function in IH. Bone marrow [<NUM>] chimeras were generated using PI3Kγ-KO and PI3Kγ-KD mice as recipients, to discriminate between kinase-dependent or kinase-independent function of PI3Kγ in the vascular compartment. The inventors introduced BM from WT into PI3Kγ-KO (WT>KO) or PI3Kγ-KD (WT>KD) animals and vice versa (KO>WT and KD>WT). After engraftment, femoral arteries were injured, and lesions were analyzed <NUM> days later. In BM WT>KO and KO>KO chimeras, the intima/media ratio was decreased by <NUM>% compared to BM WT>WT chimera (<FIG>), whereas no difference was observed in BM WT>KD chimeras as previously observed [<NUM>]. These results indicated that the presence of PI3Kγ, and not its catalytic activity, is required in the vascular compartment to induce a fibroproliferative response to the injury.

Consistently with the inventors' previous results [<NUM>], BM KO>WT, BM KD>WT or BM KD>KD chimeras showed a large decrease in intima/media ratio confirming the role of PI3Kγ catalytic activity in the immune compartment (<FIG>). Histological analysis by Masson Trichrome staining of the injured arteries showed a decreased neointima area in BM WT>KO and BM KO>WT chimeras compared to controls (<FIG>). This staining clearly indicated that the reduced neointima area in BM WT>KO is mostly due to a lower expansion of the VSMC layer (<FIG>). Taken together, these results demonstrate that the post-injury VSMC proliferation is independent from the catalytic function of the PI3Kγ but rather involves its docking function.

Given the importance of cAMP in the inhibition of VSMC proliferation in vitro and in vivo [<NUM>-<NUM>], the inventors hypothesized that a kinase-independent function of PI3Kγ could also be implicated in VSMC proliferation via cAMP regulation. To test this hypothesis, the proliferation of primary VSMC from different genotypes (WT, PI3Kγ-KO or PI3Kγ-KD) was evaluated by stimulation with a powerful mitogenic agent for VSMC, platelet derived growth factor (PDGF). An adenylate cyclase activator, forskolin (FSK), was supplemented to culture media to increase cAMP levels and counterbalance the VSMC proliferation induced by PDGF (<FIG>). The inventors' results showed that <NUM> of FSK decreased VSMC proliferation and showed that FSK effect is significantly potentiated in the absence of PI3Kγ (PI3Kγ-KO) but not in PI3Kγ-KD VSMC, confirming the role of AKAP function of PI3Kγ in VSMC (<FIG>). These results demonstrated that PI3Kγ controls VSMC proliferation by regulating intracellular concentration of cAMP, independently from its enzymatic activity.

The involvement of the PI3Kγ in controlling intracellular concentration of cAMP in VSMC was confirmed by studying the dynamic of cAMP signals in WT, PI3Kγ-KD or PI3Kγ-KO primary VSMC using the FRET-based TEpacVV biosensor [<NUM>]. Relative changes in cAMP levels in response to <NUM> forskolin were monitored over time, by calculating the ratio of donor fluorescence (F480nm) to acceptor fluorescence (F535nm). cAMP level is determined as a percentage of the maximum ratio change (Rmax). Rmax was determined at the end of the experiment by adding <NUM> <NUM>-isobutyl-<NUM>-methylxanthine (IBMX), a pan-PDE inhibitor, in the presence of <NUM> forskolin. As shown in <FIG>, cAMP production in response to <NUM> forskolin is increased in PI3Kγ-KO compared to WT primary VSMC by <NUM> folds (<NUM>% vs <NUM>% of IBMX response, p=<NUM>), while forskolin response of PI3Kγ-KD cells remains similar to WT (<FIG>). cAMP basal levels were not altered at steady states in all three genotypes (<FIG>). Moreover, the maximum ratio change was also identical in VSMC from all genotypes (<FIG>). Altogether, these results showed that the cAMP response to forskolin is higher in PI3Kγ-KO VSMC than in WT and PI3Kγ-KD cells, demonstrating a role for PI3Kγ in cAMP level limitation.

The intracellular level of cAMP depends on its production and degradation by adenylyl cyclases (AC) and PDE, respectively. PI3Kγ has already been shown to bind PDE in cardiomyocytes [<NUM>, <NUM>], and among these the inventors focused on PDE3 and <NUM> since they have been implicated in IH [<NUM>, <NUM>, <NUM>]. They successively treated forskolin-stimulated VSMC with cilostamide and rolipram, inhibiting PDE3 and PDE4 respectively, and quantified cAMP level according to the Rmax. These experiments allowed to evaluate the relative proportion of cAMP pools regulated by each PDE in different genotypes. The results showed that cAMP levels are mostly regulated by PDE4 in WT and PI3Kγ-KD cells (<NUM>% and <NUM>% respectively relatively to the IBMX response). PDE3 implication in cAMP levels regulation in these cells is limited to <NUM>% and <NUM>% of IBMX response, respectively (<FIG>, B and C). Accordingly, in PI3Kγ-KO cells the major PDE isoform degrading cAMP is PDE3 (<NUM> % of IBMX response for PDE3 vs <NUM>% of IBMX response for PDE4, <FIG>). To confirm the implication of PDE4 enzymes in cAMP regulation downstream of PI3Kγ, the inventors performed immunoprecipitation and measured PDE4 activity. They focused on PDE4D which is the main isoform expressed in VSMC. Consistent with inventors' cAMP measurements in living cells, PDE4D activity was significantly reduced in PI3Kγ-KO cells compared to WT and PI3Kγ-KD (<FIG> and E).

PI3Kγ has been shown to regulate cAMP levels and signaling via its AKAP function in cardiomyocytes [<NUM>]. Interestingly, the interaction between PI3Kγ and the PKA/PDE complex has been mapped to the N-terminal region of p110γ between amino acid <NUM> and <NUM> [<NUM>]. To further investigate the functional relationship between PI3Kγ, cAMP regulation by PKA/PDE and VSMC proliferation, the inventors disrupted the PI3Kγ/PKA interaction by treating the cells with a cell penetrating competing peptide corresponding to the binding region of PKA on p110γ, and probed for their proliferation in response to PDGF and forskolin [<NUM>]. A control peptide has been designed with two-point mutations (K>A and R>A at position <NUM> and <NUM> of the PI3Kγ sequence, respectively) that does not interact with PKA [<NUM>] (<FIG>). First, the inventors assessed the ability of the peptide to selectively interfere with the scaffold and not the catalytic function of PI3Kγ. They found that C5a-mediated Akt phosphorylation in macrophages, an event relying on the catalytic activity of PI3Kγ [<NUM>], was unaltered in cells treated with the PI3Kγ peptide, while significantly reduced in those exposed to the canonical PI3Kγ kinase inhibitor AS-<NUM> (<FIG>). Next, they determined the time course of peptide penetration in VSMC using a FITC-tagged version of the peptide. The peptide was detectable in cells after <NUM> hours and was still detectable <NUM> after incubation (<FIG>). Thus, the inventors used the PI3Kγ peptide to block PI3Kγ-PKA interaction in an experiment of cell proliferation as in <FIG>. Incubation with the blocking peptide inhibited cell proliferation in WT and PI3Kγ-KD cells (<FIG>) whereas no difference was observed in PI3Kγ-KO cells (<FIG>). These data clearly demonstrate that PI3Kγ controls VSMC proliferation via its AKAP function.

Altogether, the present results allow to conclude that PI3Kγ acts as a catalyst of VSMC proliferation by decreasing cAMP levels mainly through the direct control of PDE4 enzymes (<FIG>).

Animals: PI3Kγ-deficient (PI3Kγ-KO) mice and mice expressing a catalytically inactive form of PI3Kγ (PI3Kγ kinase-dead mice; PI3Kγ-KD) were from a C57BL/6J background and have been described earlier [<NUM>-<NUM>]. WT, PI3Kγ-KD, PI3Kγ-KO were maintained at the animal facility of Rangueil (UMS <NUM>; Anexplo platform) and kept under SPF conditions. All animal procedures were conducted in accordance with institutional guidelines on Animal Experimentation and were under a French Ministry of Agriculture license.

Femoral artery wire injury in mice : WT, PI3Kγ-KD, PI3Kγ-KO male mice aged <NUM>-<NUM> week were investigated using an established model of femoral artery wire injury [<NUM>], for IH studies. In brief, general anesthesia was achieved with <NUM>% isoflurane. Then, the femoral artery was isolated, and an incision was made under a surgical microscope (Carl Zeiss). A <NUM>-mm diameter angioplasty guide wire with a <NUM>-mm tip was advanced three times into the artery up to the level of the aortic bifurcation then pulled back. After removal of the wire, the arteriotomy site was ligated. Mice were sacrificed <NUM> days after the injury for histological and immunohistochemical analysis.

Tissue processing and morphometry: IH quantification was performed by the paraffin-embedment technique. At the point of euthanasia, mice were perfused with PBS, followed by <NUM>% paraformaldehyde. Vessels were harvested and fixed in <NUM>% formalin, pH = <NUM>, for <NUM>. They were put into paraffin and prepared as slides (<NUM>-µm-thick sections). To quantify the rate of IH, sections were stained with Masson Trichrome (Bio Optica), and then the lumen, IEL (internal elastic lamina), and EEL (external elastic lamina) perimeters (µm) were measured at <NUM> sections (<NUM>, <NUM>, <NUM>, and <NUM> from the ligation site) for each vessel using LAS software (Leica). Arteries that underwent an occlusive thrombotic event were excluded from the quantification group. The area defined by the EEL (µm<NUM>) was calculated assuming circular geometry in vivo: (AEEL = EEL circumference<NUM> ÷ 4π). The area defined by the IEL (µm<NUM>) was calculated using: (AIEL= IEL circumference<NUM>÷ 4π). The lumen area (µm<NUM>) was calculated using: (ALUM = Lumen circumference<NUM>÷ 4π). The neointimal area (µm<NUM>) was calculated using: (ANEO = AIEL - ALUM). The medial area (µm<NUM>) was calculated using: (AMED = AEEL - AIEL). The intima/media ratio was calculated using: (neointima/media = ANEO ÷ AMED).

Bone Marrow (BM) transplantation: BM was obtained from <NUM>-week-old WT, PI3Kγ-KD and PI3Kγ-KO mice. BM cells were flushed from femurs and tibias, then washed, filtered, counted, and resuspended in sterile PBS for retro-orbital injection of <NUM><NUM> unfractionated cells per WT, PI3Kγ-KD or PI3Kγ-KO irradiated mouse (<NUM> Gy; Gilardoni RadGil irradiator). <NUM> weeks later, femoral artery injury was performed, and mice were euthanized <NUM> d after the surgery. The successful engraftment was confirmed by PCR.

Isolation of SMC from murine aorta and culture: Mouse VSMCs were isolated from WT mice according to a modified protocol described by [<NUM>]. In brief, aorta from <NUM> WT mice were dissected from their origin to the iliac bifurcation, flushed with PBS, and removed. The adventitia was removed from the aorta and the smooth tubes were cut into pieces of <NUM>-<NUM>, and then digested in <NUM>% collagenase solution. Collagenase digestion was stopped by adding DMEM (D0822, Sigma Aldrich) /<NUM>% FBS (FBS, Thermo Fisher Scientific). Then, the pieces of aorta were washed twice and seeded in culture dishes. Primary confluent cultures were trypsinized (<NUM>% trypsin; Thermo Fisher Scientific) at <NUM>, and then cells were incubated at <NUM> in <NUM>% CO<NUM> in DMEM/<NUM>% FBS and cultured until the fourth passage.

Measure of mouse SMC proliferation: Primary mouse SMCs were sub-cultured onto <NUM>-well cell culture plates in complete medium. Cells were treated or not with PDGF (<NUM>-BB-<NUM>, R&D Systems) and forskolin (F3917, Sigma Aldrich) for <NUM>. For blocking peptide experiments, cells were incubated <NUM> with <NUM> of control (RQIKIWFQNRRMKWKKGAATHASPGQIHLVQRHPPSEESQAF - SEQ ID No.: <NUM>) or blocking peptide (RQIKIWFQNRRMKWKKGKATHRSPGQIHLVQRHPPSEESQAF - SEQ ID No.: <NUM>). Peptides were synthesized by GenScript (GenScript, Piscataway, NJ). BrdU (<NUM>-<NUM>, Invitrogen) incorporation assays were performed by classical immunofluorescence procedure using anti-BrdU antibody (<NUM>-<NUM>, eBioscience) and DAPI. Proliferation rate is measured as the ratio of BrdU positive nuclei over total nuclei and expressed as fold increase.

Measurement of cAMP dynamics: Primary SMC were seeded on coverslips and infected with the TEpacVV-encoding type-<NUM> adenovirus (~<NUM> particles per SMC) as described elsewhere [<NUM>]. Coverslips were placed in a microscope chamber continually perfused (<NUM>·min-<NUM>) with BBS buffer (<NUM> NaCl, <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, <NUM> NaH<NUM>PO<NUM>, <NUM> NaHCO<NUM>, <NUM> glucose) saturated with <NUM>% O<NUM>-<NUM>% CO<NUM>. Ratiometric analyses were performed as follows: fluorescence was excited with a LED source at <NUM> and fluorescence emission was monitored with a dichroic mirror (T450LPXR) and alternating emission filters for the donor (HQ480/<NUM>) and acceptor (D535/<NUM>). Pairs of images were recorded with an Orca-ER CCD camera (Hamamatsu Photonics, Japan), at <NUM>-s intervals. Changes in [cAMP]i are expressed as the ratio of donor fluorescence (F480) to acceptor fluorescence (F535). The ratios were multiplied by a same constant for all experiments, such that the baseline ratio was <NUM> in basal conditions. The maximum ratio change (Rmax) was obtained by stimulating cells with <NUM> forskolin and <NUM> IBMX (I5879, Sigma Aldrich). Filters and mirrors were obtained from AHF analysentechnik AG, Tübingen, Germany.

Measurement of PDE activity: Cells were scraped in <NUM> mmol/L NaCl, <NUM> mmol/L Tris-HCl (pH <NUM>) and <NUM>% Triton X-<NUM>, supplemented with protease and phosphatase inhibitors and centrifugated at <NUM> rpm for <NUM> at <NUM>. Supernatants were subjected to immunoprecipitation and then assayed for PDE activity or Western Blotting. For immunoprecipitation assays, <NUM>µg of pre-cleared extracts were incubated with 20µl of a <NUM>:<NUM> slurry of protein A- or protein G-Sepharose (Amersham Biosciences, Buckinghamshire, UK) and 1µg of anti-PDE4D antibody (Abcam #ab171750) for <NUM> at <NUM>. Immunocomplexes were then extensively washed with lysis buffer and subjected to PDE activity assay. PDE activity in immunoprecipitates was measured according to the two-step method of Thompson and Appleman with minor modifications. In brief, immunoprecipitations were assayed in a total volume of <NUM>µl of reaction mixture containing <NUM> mmol/L Tris-HCl (pH <NUM>), <NUM> mmol/L MgCl<NUM>, <NUM> mmol/L <NUM>-mercapto-ethanol, 1µmol/L cAMP (Sigma-Aldrich, Saint Louis, MO) and <NUM>µCi of [<NUM>H]cAMP (Amersham Bioscience, Buckinghamshire, UK) for <NUM> at <NUM>. To stop the reaction samples were boiled at <NUM> for <NUM>. The PDE reaction product <NUM>'-AMP was then hydrolyzed by incubation of the assay mixture with <NUM>µg of Crotalus Atrox snake venom for <NUM> at <NUM> (Sigma-Aldrich, Saint Louis, MO). The resulting adenosine was separated by anion exchange chromatography using <NUM>µl of a <NUM>% (w/v) suspension of Dowex AG1-X8 resin (Bio-Rad, Segrate, Milano, Italy). The amount of radiolabelled adenosine in the supernatant was quantitated by scintillation counting (Ultima Gold scintillation liquid from Perkin Elmer, Waltham, MA).

Claim 1:
A fusion peptide for use in treating, preventing and/or delaying onset of a fibroproliferative vascular disease, wherein the fusion peptide comprises:
(a) an amino acid sequence as defined in SEQ ID No.: <NUM> or a related homolog having at least <NUM>% identity with SEQ ID No.: <NUM> and having the ability of the sequence SEQ ID No.: <NUM> to inhibit the kinase-independent function of PI3Kγ, and
(b) a peptide having the ability to penetrate a cell.