Cyclic arginine-glycine-aspartate attenuates acute lung injury in mice after intestinal ischemia/reperfusion

Intestinal ischemia/reperfusion (I/R) injury is a critical problem resulting in high mortality rates of up to 60 to 80% [1‐3]. I/R causes gut barrier disruption and endotoxin entry into the circulation, leading to severe systemic inflammation and eventually multiple organ failure [4‐7]. Acute lung injury (ALI) is a common complication that occurs after intestinal I/R and is a major contributor to the high mortality rate [7‐11]. Limited pharmacological treatment options exist for ALI, with most targeting inflammatory mediators and oxidative stress pathways [12]. There is an urgent need for an effective approach for ALI treatment.

ALI induced by intestinal I/R is caused by an excessive systemic inflammatory response, triggered by the release of proinflammatory cytokines and bacteria-derived endotoxins from the reperfused ischemic gut tissue [13‐15]. Pathophysiologically, ALI is associated with the influx and activation of immune cells. With the release of abundant cytokines and chemokines, ALI can be further complicated by infection and ventilation-induced injury [13, 16]. Neutrophils are the earliest immune cells to be recruited to the site of injury or infection. Moreover, recruitment of neutrophils to the lungs is known to play a key role in the progression of ALI [17]. However, neutrophil migration into the lungs is not sufficient to cause ALI, but rather activation of neutrophils is necessary to trigger the injury [17]. Under normal conditions, neutrophils roll along microvascular walls. After activation by proinflammatory cytokines and chemotactic factors, neutrophils are then able to penetrate the vasculature and transmigrate through the interstitium into the alveolar space [18, 19]. In addition, alveolar macrophages are also essential players in the initiation of ALI [11, 20].

Integrins expressing on the cell surface mediate the interactions of neutrophils with endothelial cells and extracellular matrix (ECM) proteins to strengthen adhesion and migration for complete infiltration [21, 22]. Twenty-four different integrins are expressed in humans, each composed of noncovalently associated α and β chains [23]. Neutrophil adhesion is commonly mediated through β₂ integrins to interact with adhesion molecules (for example, intercellular adhesion molecule (ICAM)-1, ICAM-2, and vascular cell adhesion molecule-1) on the endothelial surface [17]. Inhibition of β₂ integrins attenuates neutrophil migration into the lungs [24]. In addition to β₂ integrins, α₄β₁ and α₅β₁ integrins also contribute to neutrophil migration by interacting with vascular cell adhesion molecule-1 during pulmonary inflammation [25]. β₁ and β₃ integrins mediate the interaction with ECM proteins, such as collagen, laminin, vitronectin and fibronectin [26]. In addition, integrins are also associated with phagocytosis, reactive oxygen species and cytokine productions [27, 28].

Through sequence analysis of ligands bound to integrins, a minimal recognition sequence in ligands containing amino acid sequence arginine-glycine-aspartate (RGD) has been well identified [26]. The RGD motif enables ligand binding to integrins and regulates cell growth, migration and survival [26]. In contrast, synthetic peptides containing the RGD sequence can compete with adhesive molecules for binding to integrins and disrupt the cellular activity mediated by integrin interaction. For example, a synthetic RGDS peptide has shown to attenuate lipopolysaccharide-induced pulmonary inflammation and to reduce the number of neutrophils infiltrating the lungs by inhibiting integrin-mediated mitogen-activated protein kinase activation [29]. Administration of cyclic RGD peptides inhibited the recruitment of macrophages and neutrophils, as well as depressing the expression of proinflammatory mediators in steatotic liver cold ischemia and reperfusion injury [30]. Another study demonstrated the effectiveness of cyclic RGD peptides in ameliorating ischemic acute renal failure in rats [31].

We have previously shown that treatment with milk fat globule-epidermal growth factor-factor 8, a protein containing an RGD motif, attenuates inflammation and ALI after intestinal I/R [6]. Based on our observations and other studies, we reasoned that RGD peptides have the capability of alleviating the injury caused by I/R. In this study, we treated mice with a cyclic RGD peptide (cRGD; RGDFV) that selectively interacts with β₃ and β₅ integrins [32]. We then evaluated its effectiveness on alleviating the ALI induced by intestinal I/R.

The lung is the most susceptible remote organ to be injured after intestinal I/R, which makes ALI a major cause for death [8, 9, 11]. Migration of activated neutrophils into the lungs is a hallmark for the progression of ALI [10], which is mainly mediated through the integrin-dependent pathway [17]. Synthetic peptides containing the RGD sequence have been demonstrated to be able to disrupt the cell-cell and cell-matrix interaction mediated by integrins, as well as to inhibit the cellular activity stimulated by integrin engagement.

In the current study, we showed that treatment with cRGD improved the integrity of tissue morphology not only locally on the gut but also remotely on the lungs after intestinal I/R. cRGD treatment effectively inhibited neutrophil infiltration to the organs, demonstrated by the reduction of MPO activity in the gut and lungs, and the number of Gr-1-positive cells in the lungs. cRGD treatment also attenuated the inflammatory response by decreasing the levels of TNF-α and IL-6 in serum, as well as IL-6 and MIP-2 in the gut and lungs. Correspondingly, the number of macrophages stained with CD11b in the lungs was reduced. Finally, the lungs of the cRGD-treated animals had less apoptotic cells, demonstrated by TUNEL staining and detection of cleaved caspase-3.

To our knowledge, this is the first study showing a protective effect of cRGD on ALI induced by intestinal I/R, although its effectiveness has also been demonstrated on ameliorating the injury in other organs that had undergone I/R [30, 31]. In steatotic liver cold ischemia followed by transplantation, administration of a cyclic RGD peptide (CRGDGWC) inhibited the recruitment of macrophages and neutrophils, as well as depressed the expression of proinflammatory mediators, inducible nitric oxide synthase and IFN-γ [30]. In addition, the expression of matrix metaloproteinase-9, a gelatinase involved in leukocyte migration within the damaged liver was inhibited by the treatment [30]. In a rat model of renal I/R, cyclic RGD peptides (RGDDFV and RGDDFLG) effectively ameliorate acute renal failure [31]. Both peptides were effective at lowering the tubular obstruction by predominantly preventing cell-cell and cell-matrix adhesions [31]. Cyclic RGDFV had a higher potential than cyclic RGDFLG in inhibiting cell-matrix adhesion, but both peptides were equipotent in disrupting cell-cell adhesion [31]. The relative affinity and specificity of the RGD peptides to other proteins have been indicated to be affected by other amino acid residues flanking the RGD motif [35]. Cyclic CRGDGWC has higher affinity for the α₅β₁ integrin [36], while cyclic RGDFV more selectively interacts with α_vβ₃ and α_vβ₅ integrins [32].

RGD peptides can be synthesized in two different structure types of linear and cyclic formats. Cyclic RGD is much more stable than that of linear RGD under normal conditions [37]. Linear RGD, such as GRGDS and GRGDNP, are rapidly metabolized in vivo and have a relatively low affinity for integrins [37]. On the contrary, linear RGD is more flexible in comparison with cyclic RGD. However, the cell permeability of linear RGD is concerning. Linear RGD induces apoptosis without any requirement for integrin-mediated cell clustering or signals. Instead, linear RGD enters cells to bind pro-caspase-3 containing a potential RGD-binding motif (aspartate-aspartate-methionine), resulting in activation of caspase-3 [38]. In contrast, our study with the treatment of cRGD showed a reduction of apoptotic cells in the lungs after intestinal I/R, suggesting that very limited cRGD in the cells interacted with pro-caspasae-3. This difference may be due to the rigid structure of cyclic peptides in comparison with the linear structures.

The mechanism of the cRGD on alleviating ALI-induced by intestinal I/R is attributed to the inhibition of neutrophils infiltrating the lungs. Over-recruitment of activated neutrophils causes excessive release of proteolytic enzymes, such as elastase and MPO, and reactive oxygen species including hydrogen peroxide and superoxide. Excessive production of these molecules causes disruption of endothelial barrier functions and promotes extravascular host tissue damage [19, 39]. Furthermore, neutrophils can produce cytokines and chemokines that enhance the acute inflammatory response [40]. Using real-time RT-PCR analysis, we detected an increased expression of several adhesive molecules, including β₁, β₂, and β₃ integrins, ICAM-1 and fibronectin, which are all involved in neutrophil transmigration after intestinal I/R. This observation further supports the rationale of targeting integrin-mediated interaction to control neutrophil infiltration into the organs.

In addition to neutrophils, a reduction of macrophages in the lungs was also observed in the cRGD-treated animals. Macrophages are the key cell types involved in the production of proinflammatory cytokines such as TNF-α and IL-6, as well as chemokines including monocyte chemoattractant protein-1, and MIP-2 [41]. MIP-2, a murine analog of IL-8, is a potent chemoattractant for neutrophil recruitment and activation [42]. By depletion of alveolar macrophages, alveolar macrophages have been indicated to play an essential role in ALI induced by intestinal and acute lung I/R [11, 20]. Furthermore, a recent study demonstrated that cyclic RGD could attenuate the TNF-α secretion from macrophages by inhibiting the ligation of α_vβ₃ integrin for NF-κB activation [43].

In this study, administration of cRGD at 5 mg/kg BW was based on a recent report showing that the linear RGDS peptide at 5 mg/kg BW significantly inhibited neutrophils infiltrating the lungs in a lipopolysaccharide-induced ALI model [29]. In addition, cyclic RGD at 4 mg/kg BW had been applied in the study of steatotic liver cold ischemia and reperfusion injury [30]. We also performed a pilot study at a dose of 10 mg/kg BW cRGD, but did not observe a noticeable difference in comparison with the 5 mg/kg BW dose. While excessive neutrophil infiltration causes organ damage, neutrophils are necessary for the killing of invaded pathogens [19, 39]. We therefore speculate that administration of cRGD at increasingly high doses may lessen its protective effect on ALI. To evaluate the overall effect of cRGD on attenuating organ injury and inhibiting the inflammatory response, administration of normal saline (vehicle) was used as a baseline for the comparison. Any random peptide sequence used as control could incur potential unidentified off-target effects. Moreover, the surgical procedure of intestinal I/R described here is a severe model. Most mice died within 20 hours after I/R in our pilot study, which makes it very difficult to study the effect of cRGD at later time points. In essence, therefore, the focus of this study was to examine the onset of ALI and monitor the underlying immunologic mechanisms at the early time points.

ALI is a common complication that occurs after intestinal I/R and contributes to its high mortality rate. Excessive neutrophil infiltration into the lungs is a hallmark of ALI. Integrin-mediated interaction plays an important role in regulating neutrophil transmigration as well as activating various cellular activities. Administration of cRGD attenuated the severity of ALI and local gut injury in mice after intestinal I/R. The numbers of neutrophils and macrophages infiltrating the lungs remarkably decreased after treatment. In addition, the levels of proinflammatory cytokines and chemokines in the circulation and organs were suppressed by cRGD treatment. Furthermore, the induction of apoptosis in the lungs after I/R was inhibited by cRGD. These results indicate that cRGD has the potential to develop as an effective therapeutic agent for treating ALI induced by intestinal I/R.