University of Zagreb School of Medicine

The prevalence of diabetes mellitus is increasing at a fast pace as a result of population ageing and sedentary lifestyle. Globally, the number of people with diabetes has more than doubled over the past three decades. The disease itself encompasses complex and chronic metabolic condition characterized by elevated blood glucose levels resulting from the impaired insulin production, resistance to insulin action, or both. Diabetic retinopathy (DR) is a major microvascular complication of diabetes mellitus and leading cause of visual impairment and blindness in working-age population in developed countries, resulting in more than 10,000 new cases of blindness per year. Despite the recent introduction of anti- vascular endothelial growth factor (VEGF) therapy, which has remarkably improved its prognosis and reduced its burdens, DR is still one of the heaviest disabling outcomes of diabetes. Thus, there remains a tremendous clinical need for additional effective treatments for patients who are refractory or who do not fully respond to current therapeutic options. The earliest changes in DR involve narrowing of the retinal arteries associated with reduced retinal hemodynamics, dysfunction of the inner retinal neurons, microaneurysm formation and increased permeability of the blood-retinal barrier (BRB) with the appearance of exudates, followed by changes in visual function. Later changes in DR include the worsening of these early microvascular changes, affecting the basement membrane of the retinal blood vessels. Capillaries degenerate and lose cells, particularly pericytes and vascular smooth muscle cells, leading to areas of microvascular non-perfusion with progressive ischemia and later with hypoxia-stimulated neovascularization. Diabetes is also a strong independent risk factor for stroke and is present in more than one third of ischemic stroke cases. Patients with type 2 diabetes have up to 4 times higher risk for stroke, and diabetic stroke survivors also have a higher risk of recurrence and a poorer prognosis compared to nondiabetics. Diabetes exacerbates the ischemic injury and hinders recovery by promoting inflammation through production of pro-inflammatory cytokines, stimulation of leukocyte infiltration, leading to apoptosis and neuronal death.
Both mentioned diabetic complications, retinopathy and stroke, are characterized by ischemic injury, ATP depletion, intracellular Ca2+ accumulation, glutamate-mediated excitotoxicity and increased generation of reactive oxygen species. Cessation of glucose and oxygen supply further leads to activation of an inflammatory response which contributes to the breakdown of the blood-brain/retinal barrier and the consequent formation of edema. Although re-oxygenation after ischemia is essential for cell survival, further damage develops during the reperfusion period. Bradykinin (BK) is suggested to be an important component of the nervous system inflammatory response and one of the key players in edema development, which further hinders the already disturbed blood flow in the damaged area, additionally increasing the size of ischemic lesion. Although all components of the kallikrein-kinin system are present in human retina and brain their exact physiological functions are still unknown. It has been shown that plasma levels of prekalikrein are 16% higher in patients with diabetes than in controls, 50% higher in patients with proliferative retinopathy, and that prekalikrein levels in the cerebrospinal fluid are significantly increased in correlation with the severity of brain edema. The BK actions are mediated through the stimulation of two subtypes of G protein coupled receptors (GPCR): the constitutive B2 receptor (B2R) and the inducible B1 receptor (B1R). The controversy stemming from the growing variety of information showing both harmful and beneficial actions of signalling pathways activated by BK needs to be elucidated carefully in a longitudinal manner. The existing studies all analyse only a single time point in this acute phase. This is, in general, one of the most evident pitfalls of preclinical studies, as the use of short time windows for drug administration contrasts longer and highly variable time windows for entry in clinical trials. The significance of the proposed research is that it represents a longitudinal multimodal approach for evaluating the bradykinin modulation of post-ischemic inflammatory response and recovery of neurological function through actions of the bradykinin type 2 receptor activation. The second specific and very important characteristic of the proposed project is the establishment of a completely new platform for multimodal preclinical research of retinal diseases, which would be the first platform of its kind in the Republic of Croatia, and would open up possibilities for novel and more comprehensive preclinical evaluation of experimental therapies with a more efficient preclinical to clinical research translation. The significance of the establishment of this new platform is in clearer translational approach, as the same platform which would be used to obtain the basic knowledge about the consequences of diabetes would be used to envisage, screen and verify the possible pathways for treatment of retinal diseases in general and for tackling a major research issue such as vision loss treatment. It is our hope that our research proposal will lead to development of novel therapeutic strategies for treatment of ischemic complications which occur in the course of diabetes and to make a step further to a more efficient translation of experimental therapies to the clinic.

Scientific objectives

The main objective of this project is to elucidate the role of bradykinin receptor type 2 (B2R) in diabetes associated ischemic injury in the mice brain and retina.

As part of this overall objective, the following specific objectives will be:

Specific Objective 1: Establishing the murine diabetic models
In order to achieve this objective, the experimental animals intended to develop type 2 diabetes will be fed on a high-fat diet (50% kcal fat diet). The control animals will be on a low-fat regimen, standard rodent chow containing 10% of kcal as fat ad libitum. Type 2 diabetes will be diagnosed in accordance with the international guidance for diagnosis of diabetes mellitus, according to the glucose concentrations after starvation and glucose tolerance testing. The animals intended to develop type 1 diabetes are heterozygous for the Akita spontaneous mutation (Ins2Akita) and will be purchased, propagated and maintained.

Specific Objective 2: Describing the role of bradykinin type 2 receptor (B2R) in acute and chronic phases of ischemic brain injury development in murine diabetic models
Our hypothesis is that in the acute phase of the ischemic injury activation of the bradykinin type 2 receptor has a detrimental immunomodulatory effect, and that in the chronic phases of the ischemic injury this activation will show a neuroprotective effect. Our previous studies along with others showed detrimental effects of bradykinin in the early stages of stroke, but the exact roles of the different bradykinin receptors have not been conclusively elucidated. Also, the downside of these studies was that they analysed only the acute phases of this modulatory effects, not taking in account chronic phase events.

Specific Objective 3: Establishing the platform for multimodal preclinical research of retinal diseases
To achieve the proposed objective a novel multimodal platform will be created to obtain basic knowledge about the consequences of ischemia underlying diabetic retinopathy. The significance of the establishment of a completely new platform for multimodal preclinical research of retinal diseases is in clear translational approach, as the same platform which would be used to obtain the basic knowledge about the consequences of diabetes would be used to envisage, screen and verify the possible pathways for treatment one of the major problem in humans – vision loss. Once characterized, this platform for in vivo imaging of retinal changes will represent a unique tool for efficient and targeted drug screening.

Specific Objective 4: Describing the role of bradykinin type 2 receptor (B2R) in the development of ischemic changes underlying diabetic retinopathy
Our hypothesis is that the bradykinin type 2 receptor activation in diabetic retinopathy exerts both detrimental immunomodulatory effects which specifically affect the microvasculature of the retina, but also show beneficial neuromodulatory effects by acting on the glutamate excitotoxicity.

Scientific project leader

MARINA RADMILOVIĆ, Ph.D., assistant professor

Department of Histology and Embryology, University of Zagreb School of Medicine, Croatia

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 Project team members

 MARIN RADMILOVIĆ, M.D.,Ph.D. student, Department of Ophthalmology, University Hospital Clinic „Sestre milosrdnice“, Croatia

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JELENA KEŽIĆ, Ph.D., Department of Histology and Embryology, University of Zagreb School of Medicine, Croatia

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HELENA JUSTIĆ, mag.biol.exp., Ph.D. student, Department of Histology and Embryology, University of Zagreb School of Medicine, Croatia

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ANJA BARIĆ, mag.biol.mol., Ph.D. student, Department of Histology and Embryology, University of Zagreb School of Medicine, Croatia

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Project collaborators

ZORAN VATAVUK, M.D., Ph.D., professor, Department of Ophthalmology, University Hospital Clinic „Sestre milosrdnice“, Croatia

DIRK WIEDERMANN, Ph.D., senior scientist, Max-Planck-Institute for Metabolism Research, Cologne, Germany

ANTJE GROSCHE, Ph.D., associate professor, Institute of Human Genetics, University of Regensburg, Germany