Roles of Kinins in the Nervous System

Abstract

The kallikrein-kinin system (KKS) is an endogenous pathway involved in many biological processes. Although primarily related to blood pressure control and inflammation, its activation goes beyond these effects. Neurogenesis and neuroprotection might be stimulated by bradykinin being of great interest for clinical applications following brain injury. This peptide is also an important player in spinal cord injury pathophysiology and recovery, in which bradykinin receptor blockers represent substantial therapeutic potential. Here, we highlight the participation of kinin receptors and especially bradykinin in mediating ischemia pathophysiology in the central and peripheral nervous systems. Moreover, we explore the recent advances on mechanistic and therapeutic targets for biological, pathological, and neural repair processes involving kinins.

Introduction

The first evidence of the kallikrein-kinin system (KKS) was reported in 1909, when Abelous and Bardier observed a reduction in the blood pressure of dogs after intravenous injection of a human urinary extract. Priban and Hernheiser showed a similar effect in rabbits, and in 1928, a hypotensive substance was detected by Frey in human urine. Two years later, Kraut called it kallikrein, which was described by Werle as a proteolytic enzyme (kininogenase) that cleaves kallidinogen (or kininogen) from blood plasma into kallidin (a kinin also known as Lys-bradykinin) [these historical events were reviewed in (10, 32)].

Rocha e Silva and coworkers described bradykinin in 1949 as another active component resulting from kallikrein activity whose precursor, bradykininogen, is found in the α2-globulin fraction of normal mammalian plasma (77). They observed a slow and delayed contraction after adding venom of Bothrops jararaca and blood into the perfusion bath containing isolated guinea pig gut. This effect was attributed to an, until that moment, unknown substance that when injected into the veins of rabbits and cats also led to a hypotensive response. Similar results were evoked by other proteolytic and coagulating snake venoms and by trypsin. Thus, the term “kinin ” is collectively used to designate polypeptides generated or released from stores in the blood or in tissues that are pharmacologically active on the smooth muscle and produce alterations in arterial tension.

In this context, the KKS includes tissue and plasma kallikreins acting on low- and high-molecular weight kininogens (LMW and HMW), respectively, to produce bioactive kinins. The half-life of these kinins is short with less than 30 s in the plasma; they are promptly hydrolyzed by the enzymatic action of zinc metallopeptidases. Briefly, the cleavage of LMW kininogen generates Lys-bradykinin (or kallidin), which is converted into bradykinin (des-Arg⁹-bradykinin) through the removal of the Arg⁹ residue from the COOH-terminal of the molecule by a plasma kininase I called carboxypeptidase N (CPN). In contrast, bradykinin is directly released by the cleavage of HMW kininogen and then metabolized into des-Arg¹⁰-Lys-bradykinin by carboxypeptidase M (CPM). Aminopeptidase P also participates in kinin degradation, and a kininase II, known as neutral endopeptidase (NEP), inactivates kinins by removing the Phe⁸-Ag⁹ dipeptide (10). However, the major degradation pathway of bradykinin involves another kininase II, angiotensin-converting enzyme (ACE). This peptidase inactivates bradykinin by sequential removal of dipeptides from its COOH-terminal, first Phe⁸-Arg⁹ and next Ser⁶-Pro⁷. ACE is also responsible for the conversion of inactive angiotensin I into the vasopressor angiotensin II. Therefore, ACE controls blood pressure by linking two different pathways, vasodepressor kallikrein-kinin and vasopressor renin-angiotensin (10, 24).

The cellular effects of kallidin and bradykinin are mediated by two bradykinin G-protein-coupled receptors: the kinin-B2 or bardykinin receptor (B2BKR), which is constitutively expressed in a wide variety of tissues and is promptly desensitized after ligand binding, and the kinin-B1 receptor (B1BKR), whose expression is low in healthy conditions but increased during inflammation, infection, or injury (51, 86). The B2BKR shows higher affinity to kinins; the B1BKR usually binds to its metabolites (14). Nonetheless, the activation of both receptors stimulates the membrane phospholipid metabolism by activating phospholipase C and promotes intracellular calcium mobilization by inositol 1,4,5-triphosphate. Secondary intracellular effects also include the release of nitric oxide (NO) and prostaglandins (51, 86).

The interrelation between renin-angiotensin and KKSs has been demonstrated in many studies, suggesting that the protective effect of ACE is partially mediated by a direct potentiation of kinin receptors due to augmented levels of circulating bradykinin. While ACE efficiently catabolizes kinins, the hypotensive response promoted by ACE inhibitors can be abolished by B2BKR antagonists [reviewed in (10, 30, 51)]. Moreover, ACE inhibitors have also shown to directly bind and activate the B1BKR in the absence of ACE and peptide ligands (34, 35). Importantly, bradykinin is implicated in many other physiological conditions besides vasodilatation, including nociception, natriuresis, increased vascular permeability, and inflammation (60).

Studies in rodents allowed the identification and localization of kallikrein, B1BKR, and B2BKR in the central nervous system (CNS) (15, 16, 27, 36, 73) and brought relevant insights on the KKS in normal and pathological physiology (49, 59, 69, 70). All these components were found in most regions of the human brain, such as thalamus, hypothalamus, cerebral cortex, and spinal cord (50, 74). Taken together, these aspects enlighten why bradykinin and the KKS have been extensively investigated for therapeutic approaches.

Bradykinin, Proliferation, and Differentiation of Neural Stem Cells

During nervous system development, neural stem and progenitor cells proliferate and differentiate in a highly accurate pattern in which these two processes are strictly correlated. Indeed, proliferation is the first stage of neurogenesis, and a specific type of cell division, termed asymmetric, predicts the differentiation of one of the daughter cells (31, 65). Next, differentiation coordinates with cell cycle exit through dual actions coming from elements of both cell cycle and differentiation machineries (4, 57, 64).

Neural stem or progenitor cells also perform symmetric division, giving rise to daughter cells with equivalent fates, typically copies of the mother cell (proliferative division). This usual type of proliferation goes in an opposite direction when compared to neural differentiation. In other words, symmetric division accounts for the expansion of the precursor cell population during nervous system development, while differentiation generates cellular diversity. During mammalian neurogenesis, neuroepithelial cells switch from proliferative to asymmetric division accompanied by or closely followed by differentiation (33, 56). In this context, there is a fine balance between proliferation and neural differentiation required for the perfect formation of the nervous system and maintenance of its homeostasis in adulthood. The balance between proliferation and neural differentiation is controlled by several classes of molecules, including products of neurogenic genes and factors in the microenvironment or niche (9, 13, 47). Recent studies demonstrate a new role for bradykinin simultaneously affecting proliferation and neurogenesis.

The tridimensional neurosphere [formed by neural precursor cells (NPCs)] is able to mimic some complex processes that occur in the early stages of neural development, such as proliferation, neurogenesis, and gliogenesis. Nonetheless, simplified models as rat adrenal pheochromocytoma (PC12) cells provide relevant knowledge regarding bradykinin action and signaling pathways. Kozlowski and coworkers demonstrated the participation of bradykinin in neural differentiation and neurite outgrowth of PC12 cells (43). Furthermore, recent studies also showed that bradykinin activates ERK1/2 via a Ca²⁺-dependent pathway involving the nonreceptor tyrosine kinases Pyk2 and Src (19), and B2BKR-mediated ERK activation requires dual signaling via both PKC pathway and EGF receptor transactivation (2). Then, this route exhibits an unquestionable role in neuronal differentiation and proliferation of undifferentiated cells (78, 79).

Martins and coworkers observed the effect of B2BKR in the formation of embryonic bodies and neuronal differentiation of murine P19 embryonal carcinoma cells. Specific inhibition of B2BKR by the antagonist HOE-140 leads to a decrease in size of embryonic bodies. Moreover, differentiated neurons exhibit a lower expression and activity of muscarinic acetylcholine receptors, suggesting a central role for B2BKR in proliferation, differentiation, and acquisition of the cholinergic fate. Since P19 cells display high rates of bradykinin release and B2BKR activity during in vitro neural differentiation, these data indicate that neural fate determination could be trigged by bradykinin (55).

Using embryonic pluripotent and multipotent stem cells, our group reported the promotion of neurogenesis by bradykinin. Briefly, this kinin increases the expression of β3-tubulin (neuronal marker), whereas GFAP and S100β (glial markers) expression is reduced. The opposite effect is observed in NPCs derived from B2BKR knockout mice (B2BKR KO) and in wild-type (WT) NPCs treated with HOE-140. Additionally, while analyses of in situ hybridization of WT mouse embryos revealed the presence of B2BKR mRNA throughout the nervous system, B2BKR KO embryos showed decreased expression of neuronal markers in several stages of development, corroborating the involvement of bradykinin in embryo neurogenesis (88).

Besides the influence in cell fate determination, it is important to highlight that treatments with bradykinin during differentiation of NPCs promote neurogenesis at the expense of proliferation. Trujillo and coworkers demonstrated that differentiation of rat neurospheres in the presence of bradykinin does not alter cell viability but results in a significantly lower proliferation rate compared to untreated differentiated cells (88). The mechanism underlying how this kinin acts on proliferation of undifferentiated NPCs is still unclear. Depending on the cellular context, bradykinin evokes opposite responses: it stimulates proliferation in quiescent cells (microenvironment without mitogenic stimulus) but suppresses cellular division in proliferating cells (microenvironment with mitogenic stimulus; the mitogenic stimuli induce proliferative division) (5, 22, 67). In this context, Dixon and coworkers verified that bradykinin suppresses platelet-derived growth factor (PDGF)-induced proliferation in muscle cells, accompanied by sustained ERK activation (20). Likewise, Duchene and coworkers demonstrated the induction of proliferation of quiescent mesangial cells by bradykinin, which could be diminished under mitogenic conditions (presence of serum) through an interaction between phosphatase SHP-2 and B2BKR (22). In view of that, bradykinin may affect cell decision to elicit or suppress proliferation in undifferentiated cells, closely followed by differentiation.

Another mechanism underlying neural cell fate determination might be proposed through gene expression analysis of NPCs, since the presence of bradykinin resulted in increased NeuroD1 (neurogenic transcription factor) and decreased Notch1 (receptor with gliogenic role) expression levels. On the other hand, reduced expression of Ngn1 (neurogenic transcription factor), eNOS, and nNOS was observed in NPCs under chronic treatment with HOE-140, together with an upregulation of Notch1 and STAT3 (gliogenic signaling pathways) expression (88).

Additionally, it is important to note that calcium signals take place in the earliest steps of nervous system development, including neural induction, neurogenesis, and neuroglial switch [for review, see (45)]. During NPC neurogenesis, B2BKR activity resembles the typical response of a G-protein-coupled receptor: intracellular free calcium [Ca²⁺]_i elevation and a subsequent decay to baseline (6, 54, 55, 88). In PC12 cells, the elevation of [Ca²⁺]_i is due to mobilization of calcium from intracellular stores and influx of this ion from the extracellular medium, which occurs through activation of voltage-independent calcium channels (6).

Bradykinin and Neuroprotection

Under normal conditions, the B2BKR accounts for the majority of the biological responses of bradykinin, and it is ubiquitously expressed in various cell types in the central and peripheral nervous systems, including neurons, astrocytes, microglia, and endothelial cells. In contrast, B1BKR is not expressed in most neural tissues, but its expression is enhanced in inflammatory events (51). Several studies pointed out that B1BKR and B2BKR can elicit inflammatory cascades. As inflammation mediators, kinins participate in edema formation by promoting vasodilatation and increased vascular permeability, in addition to immune cell invasion and stimulation of cytokine release and other compounds, such as prostaglandins and leukotrienes (10, 29, 72, 83, 87, 100). Therefore, the intricate enzymatic cascade system, the broad range of action, and the wide expression of the KKS components in the nervous system bring our attention to the physiological role of the bradykinin and its receptors in the brain, especially under cerebral injury conditions. Bradykinin also participates in important physiological responses after cerebral injury, such as blood-brain barrier (BBB) disruption and neuroprotection.

Neuroprotection aims to diminish the molecular and cellular damages that occur in ischemic injury, preventing or attenuating the progression of the disease and its secondary consequences. Similarities are observed in many biochemical mechanisms underlying the neurodegenerative process associated with CNS injuries. Increased activity of glutamatergic receptors, accumulation of an excess of intracellular calcium, oxidative stress, inflammatory response, and apoptosis are the key pathways in the progression of ischemic damage, especially in the penumbral zone (28, 38, 81).

The neuroprotective properties of the KKS, especially bradykinin, in the ischemic brain started to be investigated in early 1990s, when Kamiya and coworkers observed an association of high levels of plasmatic and tissue bradykinin with the progression of cerebral edema. The authors introduced the molecular basis to modulate the KKS in order to effectively treat human stroke and potentially find synergistic drugs to attenuate secondary injury mechanisms: the increase of ischemic edema, the ATP loss, and the lactate accumulation could be suppressed by inhibiting bradykinin synthesis (39, 40). However, only almost one decade later, the mechanism of KKS started to be unveiled. Using adenovirus gene delivery, Chao's research group showed that kallikrein/kinins after myocardial ischemia are able to decrease apoptosis through the Akt signaling pathway (98, 99).

In the CNS, the kallikrein gene transfer technique was used to promote neuroprotection against stroke (93). Following the obstruction of the middle cerebral artery, ventricular injections of adenovirus expressing the human tissue kallikrein gene significantly enhanced the survival and migration of glia cells into the penumbra and core zones. The overexpression of kallikrein gene also brought apoptosis near to control levels by increasing the production of cerebral NO, pAkt, and Bcl-2, and reducing oxidative stress and caspase 3 activation. More importantly, both migration and apoptosis effects were abolished in the presence of a B2BKR-specific antagonist. In this context, Noda and coworkers showed that besides astrocytes and oligodendrocytes, microglia also expresses functional kinin receptors that can activate and induce cytokine release (62). B2BKR are expressed in nonactivated microglia; activated microglia mainly expresses B1BKR. Additionally, the same research group demonstrated that bradykinin has a protective role in the CNS by reducing lipopolysaccharide inflammation and neuronal death in neuron-microglia cocultures (63). These findings suggest a major play of the KKS in glial cells, especially in the control of CNS inflammatory responses.

Besides the effect on glia cells and inflammation, kallikrein can boost angiogenesis and promote neurogenesis after ischemia (91). Similarly to previous studies, kallikrein gene transfer technology was used to investigate the importance of the KKS in a delayed systemic administration after injury. Kallikrein overexpression reduced neuronal apoptosis and inhibited inflammatory cell accumulation, but these effects were abolished by blocking B2BKR. Moreover, B2BKR activity was able to promote angiogenesis and neurogenesis after ischemia/reperfusion injury, indicating for the first time that the B2BKR could elicit neurogenesis.

The importance of B2BKR as a major player in the protective effect of bradykinin was highlighted with the introduction of the B2BKR KO mouse model (11). In 2006, Xia and coworkers reported an increase in the mortality rate, in infarcted areas, and in neurological deficit scores after 2 weeks of ischemic brain injury in B2BKR KO mice (92). These results indicate that the B2BKR stimulates survival and protects against brain injury by suppression of apoptosis and inflammation. Two years later, the validity of the findings was questioned, since the infarctions areas could ultimately result from insufficient vessel occlusion or other technical limitations (42). Even facing some contradictory data, bradykinin and, especially, B2BKR are well known for controlling the postischemic BBB disruption and alleviation of ischemic injury progression in a specific time window (41, 76, 83). However, growing evidence puts also B1BKR into the spotlight as a coplayer or major agent in the formation of brain edema (3, 7, 82).

In 2009, Austinat and coworkers reported that B1BKR KO mice have smaller brain infarctions and less neurological deficits with reduced postischemic inflammation (7). The involvement of the B1BKR in ischemic injury was also confirmed by pharmacological assays in which B1BKR inhibition by R-715 recovered the ischemic tissue in a dose-dependent manner. Likewise, Albert-Weissenberger and Raslan, with their respective collaborators, reported that B1BKR blockage protects against cortical cryolesion by reducing inflammation and edema formation, suggesting that astrocyte activation, reduction of axonal injury and BBB leakage are the main underlying mechanisms (3, 76).

Bradykinin has also been associated with astrocytic glutamate release and N-methyl-d-aspartate (NMDA) receptor-mediated increase in neuronal intracellular calcium concentration and glutamate neurotoxicity (12). Yasuyoshi and coworkers exposed rat retina primary cultures to an excess of glutamate and verified the cellular viability in the presence and absence of bradykinin. With this simple methodology, the authors observed bradykinin-induced protection against glutamate neurotoxicity associated with NO and oxygen radical generation (97). Recently, Martins and coworkers claimed that the B2BKR and B1BKR are related to NMDA excitotoxicity in rat hippocampal slices. B2BKR neuroprotection after NMDA-induced excitotoxicity was decreased in the presence of HOE-140. However, bradykinin-promoted neuroprotection was abolished in the presence of a B1BKR agonist (53).

Bradykinin in Pathophysiology and Repair of Spinal Cord Injury

To date, very limited work has been done to explore the roles of bradykinin in pathophysiology, neuroprotection, and recovery neurobiology of spinal cord injury (SCI). PubMed^® showed 39 papers after a search using combinatorial key words of “bradykinin and spinal cord injury ” (performed on March 2, 2015), among which only eight reports actually have SCI in the title or abstract. Thus, this section aims to present an introductory description about work conducted so far to investigate the multifaceted effects and prospects of bradykinin on pathophysiology (e.g., inflammation, autoimmune, neuronal degeneration, blood-spinal cord barrier, hyperalgesia, etc.), neuroplasticity, neural protection, and neural recovery after SCI or spinal cord-related neural injury.

Acute Pathology, Inflammation, and Autoimmune Reactions After SCI: Roles of Bradykinin

Early studies revealed that, similar to peripheral tissue injury, activation of the KKS took place after experimental SCI in rat, which suggested that bradykinin might also contribute to the pathogenesis of vasogenic neural edema observed in posttraumatic brain vascular injury. Specifically, it was determined that kininogen content in injured spinal cord epicenter segment increased in a time-dependent manner (an increase up to 40-fold over 2 h following SCI), and in vitro trypsin treatment of the kininogen in traumatized spinal cord resulted in production of kinins, as detected by HPLC and quantified with a radioimmunoassay. These findings suggest that after SCI there is an increased accumulation of kininogen, and its conversion to vasoactive kinins by kallikrein plays potential roles in the secondary injury process, pathophysiological complications, and/or neural repair (94). Indeed, bradykinin is one of the first biological compounds generated at the lesion site of spinal cord parenchymal tissue, and it can initiate a cascade of inflammatory events typical for traumatic, ischemic, or hemorrhagic SCI.

For acute outcomes triggered by primary lesion insults, bradykinin and other kinins are shown to induce pathological changes in subarachnoid and intraparenchymal hemorrhage and secondary ischemia (26). Conversely, in terms of posttrauma edema, Xu and coworkers investigated whether bradykinin could regulate the expression of aquaporin-4 (AQP4) proteins in a rat transient spinal cord ischemia model (95). The study revealed that the expression of AQP4 protein after spinal cord ischemia/reperfusion was a tissue compartment-specific and time-dependent event; preexposure to bradykinin could attenuate AQP4 expression in white matter close to the lesion side. These results indicate a possible role of bradykinin in ameliorating spinal cord ischemic edema (95).

One of the profound pathological consequences of SCI is breakdown of the blood-spinal cord barrier, which further exacerbates inflammation and other secondary injury processes by allowance of blood-borne proinflammatory factors, such as tumor necrosis factor-α (TNF-α), to enter the spinal cord interstitial space. Bradykinin has been determined as one of the potent molecules that cause the biphasic disruptions of the blood-spinal cord barrier after SCI. Interestingly, pretreatment of the decapeptide B9430, a potent bradykinin antagonist, decreased the general blood-spinal cord barrier disruption occurring immediately after SCI but did not affect the delayed opening of the blood-spinal cord barrier evaluated at 72 h after injury (66).

Disruption of the blood-spinal cord barrier and transendothelial trafficking of immune cells into the CNS is observed after SCI in multiple sclerosis (MS) and in other neurological conditions (29). Using mouse models of experimental autoimmune encephalomyelitis (EAE), investigators reported that kinins increased vascular permeability and enhanced inflammation by acting on distinct bradykinin receptors. Following myelin oligodendrocyte glycoprotein [MOG(35-55)]-induced EAE, B1BKR, but not B2BKR expression, was markedly augmented in inflammatory CNS lesions in mice. However, the EAE pathology level was significantly attenuated in B1BKR KO mice compared with WT controls, and treatment of WT mice with the B1BKR antagonist R-715 before and after EAE onset showed nearly equal efficacy, but B1BKR activation by R-838 further worsened EAE pathology. Notably, B1BKR (not B2BKR) inhibition could markedly reduce BBB permeability by reversing upregulation of intracellular and vascular cell adhesion molecules (ICAM-I and VCAM-I) at the inflamed BBB and thereby limiting T-cell transmigration (29). These results collectively suggest that there may be good therapeutic value for developing more effective bradykinin antagonists in order to block post-SCI inflammatory damage resulting from blood-spinal cord barrier disruption. However, research endeavors with more diversified lesion models of SCI and more precise cell type targets appear to be needed in future studies to better define specific profiles of bradykinin actions in SCI-related secondary injury etiology and repair mechanisms.

Pathogenic Contribution of Bradykinin to the Development of Neuropathic Pain After SCI

Neuropathic pain is a leading debilitating morbidity resulting from SCI, and it remains an unmet medical demand. In a rat model of contusion SCI, more than twofold increase was observed for the expression of B1BKR and vanilloid-1 (TRPV-1) receptor genes in the injured segment dorsal horn region of the spinal cord in rats manifesting hyperalgesia behavior compared with SCI rats that did not show hyperalgesia (21). It turned out that the spinal cord B1BKR and B2BKR gene expression levels can also be promoted by peripheral nerve injury, and the increases take place in the dorsal root ganglia (DRG) neurons as well. Administration of selective nonpeptide antagonists of B1BKR (LF22-0542) and B2BKR (LF16-0687) can ameliorate thermal hyperalgesia behaviors after partial ligation of the sciatic nerve, yet both antagonists failed to block tactile and cold allodynia, implying the role of both kinin receptors in inducing thermal hyperalgesia but not in tactile and cold allodynia associated with peripheral nerve injury (71).

Indeed, in another study, treatment with AMG9810 (TRPV-1 antagonist) improved thermal hyperalgesia likely as a result of a generalized analgesic effect, whereas the effect of Lys-(des-Arg⁹, Leu⁸)-bradykinin, a B1BKR antagonist, appears to work to specifically reverse thermal hyperalgesia in a rat contusion model of SCI (75). However, the effect of B1BKR blockers to prevent a particular type of neuropathic pain could be a lesion pathology designated event. For example, partial ligation of the sciatic nerve causes a severe and long-lasting reduction in mechanical and thermal nociceptive thresholds (i.e., mechanical and thermal allodynia) in the paw ipsilateral to nerve lesion in the WT mouse; B1BKR KO resulted in a significant decrease in early stages of mechanical allodynia and thermal hyperalgesia. Moreover, systemic treatment with the B1BKR selective antagonist des-Arg⁹-[Leu⁸]-bradykinin reduced the established mechanical allodynia observed 7-28 days after nerve lesion in WT mice (25). Taken together, current data suggests that bradykinin receptor blockers hold substantial therapeutic potential for the treatment of some aspects of neuropathic pain post-SCI and upcoming studies should look into how to develop more effective specific antagonists of B1BKR and B2BKR that are suitable for clinical applications.

The Role of Neuroprotection and Neurogenesis of Bradykinin for Investigating and Treating SCI

Recently, it has been increasingly appreciated that bradykinin may also act as a “double-edged sword, ” a feature that is shared by many inflammatory mediators. Besides the aforedescribed proinflammatory, proneural degeneration and pain pathogenic effects, bradykinin, depending on its action-mediating receptor subtype as well as effect intensity, time course, and loci, can provide prorepair and even neurogenic impacts for the lesioned spinal cord. Even for the process of post-SCI establishment of hyperalgesia, bradykinin, in addition to playing a role of a central inflammation mediator, was shown to be likely involved in triggering expression increase of GABA_A receptors in DRG neurons underlying a neural process referred to as dorsal root reflex (DRR). DRR describes action potential initiated following acute spinal cord parenchymal injury in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery to cause hyperpolarization and presynaptic inhibition in order to impede pathology-elevated release of neurotransmitters (46, 90).

Because of the multimodal actions of bradykinin, investigators performed bradykinin preconditioning experiments to examine its potential neural protective effects on the spinal cord in vivo. Based on data generated from a rat spinal cord ischemic injury study, it was reported that IV administration of bradykinin 15 min before ischemia significantly promoted hindlimb locomotor scores assessed using Tarlov's scale. The beneficial effect was probably derived from better preserved integrity of the blood-spinal cord barrier that could be also improved by treatment of B9430, a B2BKR antagonist, and augmented expression of basic fibroblast growth factor (bFGF) protein for neuronal protection (84, 96). Moreover, in a rabbit model, IP bradykinin preconditioning at 48 h before 20 min of abdominal aorta ligation resulted in significantly improved group average Tarlov score for hindlimb locomotion; the functional benefits might result from significantly decreased activities of total superoxide dismutase (SOD), mitochondrial Mn-SOD, CuZn-SOD, and catalase activities in the bradykinin-preconditioned groups compared with the ischemic control group. These results suggest a possibility of recapitulating the neural protective effects of bradykinin precondition via devising drug strategies for mitochondrial protection and reduction of Mn-SOD synthesis (58).

Other bradykinin-oriented neural protection regimens directly aim to develop kinin receptor antagonists into neuroprotective agents for neurotrauma, including SCI. Interestingly, present drug development is primarily interested in establishing agents to block activation of bradykinin and substance P receptors since the two neuropeptides are known to set neurogenic inflammation in motion, resulting in vasodilation, plasma extravasation, and the subsequent neural edema, all key players in the progressive secondary injury that determines severity of chronic neurological deficits. Thus, this strategy is focused on alleviating acute pathology for SCI, traumatic brain injury, and stroke (86). Furthermore, investigators reported that dynorphin A (2-17), a postcleavage endogenous opioid peptide derived from the dynorphin (1-17), can bind to B2BKR to facilitate the response of wide dynamic range neurons to innocuous and noxious mechanical stimuli in rats with neuropathic hyperalgesia, suggesting that blocking spinal bradykinin receptors may become a new therapeutic option for treating chronic neuropathic pain (8).

Importantly, the neural recovery potential of bradykinin for SCI and other forms of neurotrauma has been lately indicated by its capability to ignite neural plasticity and neurogenesis. Trujillo and coworkers reported a novel function of bradykinin and B2BKR interaction that promotes neuronal differentiation of neural stem cells (NSCs) (88). Briefly, by applying the B2BKR antagonist HOE-140, they found that rat neurosphere under differentiation drive reduced expression of neuron-specific β3-tubulin and enolase with corresponding increase in GFAP expression. Their data shows that bradykinin-induced receptor activity contributes to neurogenesis. Consequently, bradykinin treatment in the middle and late stages of the differentiation process augmented neurogenesis (88). This report has added another important piece of evidence that proinflammatory factors can play an important role in post-SCI onset of neurogenesis that has established impact on neural plasticity and functional recovery. Such prorepair effects are empowered by the functional multipotency of NSCs that, via secreting trophic factors and prohealing cytokines, promote neuroprotection, serotonergic neurite outgrowth, and synaptic reorganization, helping engender functional recovery after SCI (37, 85).

In summary, investigation of different effects of bradykinin on SCI pathophysiology and recovery neurobiology already demonstrates serious potential of delegating effective basic science, translational studies, and even clinical applications of bradykinin-related tactics for advancing fundamental understanding and therapeutic development for SCI and other neurotrauma conditions. Clearly, future studies can benefit from designing hypothesis-driven projects that explore specific mechanistic and therapeutic targets for post-SCI biological, pathological, and neural repair processes involving bradykinin.

Conclusions and Perspectives

The functions of bradykinin and B2BKR in brain injury and neurodegeneration are controversially discussed. Kinins have been attributed with functions in stroke, Alzheimer's disease, multiple sclerosis, and epilepsy (17, 18, 68, 80). Bradykinin-mediated actions are not limited to arterial pressure control and inflammation. Besides participation in neurotransmission, developmental functions are associated with this neuropeptide in favoring neurogenesis over gliogenesis (Fig. 1).

Figure 1.

The role of bradykinin in neuroprotection and neurogenesis. The activation of B2BKR promotes neurogenesis, while its blockage stimulates gliogenesis and is related to an increase in postischemic injury in KO animals. Failure in neuroprotection is also observed following B1BKR activation. Blockage of B1BKR, inhibition of bradykinin production, and overexpression of the kallikrein gene alleviates brain injury. B2BKR and B1BKR are related to NMDA excitotoxicity: B2BKR antagonists decrease neuroprotection after NMDA-induced excitotoxicity, which is promoted by B1BKR agonists.

ACE inhibitors have come into focus in neuroscience, due to their benefits in neuronal diseases. Although underlying mechanisms may involve modulation of angiotensin receptor activity through blocking conversion of angiotensin I into angiotensin II, the augmented half-time of bioactive kinins is responsible at least in part for the observed neuroprotective effects. In vitro and in vivo studies support this affirmation, showing that bradykinin is neuroprotective in ischemic stroke. In view of that, the proposed disease-deteriorating actions of bradykinin and its increased concentration in the cerebrospinal fluid correlate with the intensity of edema formation in patients suffering from traumatic brain injury or stroke (61). As a further indication, disease progression in B1BKR KO mice shows functional improvement compared to WT animals following traumatic brain damage (7). Neuroprotective functions of bradykinin were also observed in animal models of temporal lobe epilepsy (68). Animals deficient in B2BKR revealed decreased survival of pyramidal cells and augmented mossy fiber sprouting, processes associated with disease development (1).

In line with such hypotheses, increased levels of bradykinin would provide a protective response for limiting further cell damage and edema, and kallikrein-6 could be used as a prognostic marker for patients with aneurysmal subarachnoid hemorrhage (52). On the other hand, edema and inflammation were less evident in kininogen-deficient mice, compared to WT animals (44). High concentrations of plasma kallikrein and an increased B2BKR activity in the cortex of Alzheimer's disease patients could indicate the participation of this receptor signaling pathway in inflammation-related processes (89). However, activity of this kinin receptor could also be related to the neurogenesis observed in the development of the disease.

Evidence for neurogenesis-promoting actions of kinin signaling come from in vitro models including neural and pluripotent stem cells (88). In vivo studies, such as the injection of bradykinin into the subventricular zone and lesion sites of brain injuries (sites of ischemia, striatum in case of Parkinson's disease) will reveal possible adult neurogenesis and neurogenerative properties promoted by the peptide. Nonetheless, further studies will need to demonstrate whether bradykinin-promoted signaling includes actions on T-cells, since T-cell mobilization has been revealed as a mechanism of hippocampal neurogenesis (48).

Stable bradykinin analogs, not subject to degradation into the B1BKR agonist des-Arg⁹-bradykinin or inactivation by ACE, may develop into therapeutic agents for neurodegenerative diseases. Labradimil (Cereport or RMP-7) is a 9-amino acid peptide designed with increased half-life in plasma that selectively activates the B2BKR. This stable molecule was shown to enhance BBB permeability, but with prolonged effects compared to bradykinin (23). Therefore, Labradimil provides an excellent tool to determine neuroprotective and neuroregenerative effects of B2BKR without any background activity of B1BKR.