**1. Introduction**

The transcription factor hypoxia inducible factor 1α (HIF-1α) is involved in vascular remodeling and angiogenesis [1]. Ischemic cardiovascular disease is characterized by reduced tissue perfusion and reduced tissue oxygen partial pressure (hypoxia), which represent a strong stimulus for HIF-1α activation [2]. HIF-1α induces the expression of several angiogenic genes, such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) or matrix metalloprotease 2 (MMP-2), which are potent inducers of angiogenesis and arteriogenesis [1,3]. It has therefore been the target of therapeutic strategies to increase tissue perfusion in ischemic limbs [4,5] and to improve wound

healing [6,7]. During normoxic conditions, HIF-1 α is hydroxylated on prolyl residues within its oxygen-dependent degradation (ODD) domain, leading to von-Hippel-Lindau protein (pVHL) dependent ubiquitinylation and subsequent degradation by the proteasome [8,9]. Hypoxia inhibits the proteasomal degradation of HIF-1 α by inhibition of the prolyl hydroxylation domain containing enzymes (PHD), resulting in stabilization of HIF-1 α and subsequent accumulation within the cell [8,9]. Additionally, it has been shown that HIF-1 α may be degraded by the protease calpain [10].

The tyrosine phosphatase SHP-2 has been demonstrated by us to positively influence angiogenesis in vitro and in vivo [11–13] and may constitute an interesting future therapeutic target within this context. In an earlier study we demonstrated that SHP-2 is important for HIF-1 α stabilisation and activity during hypoxia, resulting in enhanced hypoxia induced HIF-1 α dependent revascularisation of wounds in vivo [12]. Further, we showed that SHP-2 activates the Src kinase upon hypoxia, which in turn influenced HIF-1 α prolyl hydroxylation [12]. Finally, we found that the impaired HIF-1 α accumulation observed upon SHP-2 inactivation could be rescued by treatment with a PHD inhibitor as well as the proteasome inhibitors MG132 and Epoxomicin [12]. However, while our data indicated that SHP-2 influences HIF-1 α accumulation during hypoxia by a ffecting the activity of the PHD, thus determining the proteasomal degradation of HIF-1 α, we did not investigate whether the e ffect may additionally be caused by regulation of proteasome activity.

The 26S proteasome is responsible for the degradation of ubiquitinylated proteins and consists of a 20S core particle and two 19S regulatory particles [14]. The 20S core particle exhibits three peptidase activities (caspase-like; C-L, trypsin-like; T-L and chymotrypsin-like; CT-L), which are responsible for the cleavage of protein substrates. Ubiquitinylated proteins are recognized and bind to the 19S regulatory particle, which is in addition responsible for the ATP-dependent unfolding of the substrate protein and the opening of the 20S core particle, where degradation occurs [14]. The activity of the 26S proteasome has been shown to be regulated by phosphorylations via threonine/serine kinases as well as tyrosine kinases on several subunits of the 19S and 20S particles [15]. However, not very much is known regarding the role of phosphatases in regulating 26S proteasome activity. Moreover, its regulation during hypoxia still needs to be investigated.

In this study, we investigated the activity of the 26S proteasome during hypoxia and the connection to SHP-2 in endothelial cells in vitro and in hypoxic wounds in vivo. We found SHP-2 to inhibit the 26S proteasome activity in hypoxic cells, as assessed by measuring 26S peptidase activity as well as the accumulation of a HIF-1-ODD-Luc reporter construct. Importantly, we demonstrated that SHP-2 regulates 26S proteasome activity in hypoxic wounds in vivo.
