Involvement of immature endothelial cells in vascular alterations in Alzheimer’s disease

Introduction
The development of neurodegenerative disorders such as Alzheimer’s disease (AD) is generally associated with a wide range of histological and pathophysiological alterations. Although the diverse triggers of the neurodegenerative processes and their interactions are still the topic of extensive debate, possible contribution of cerebrovascular deficiencies has been suggested in the recent years. Alzheimer’s disease has emerged as one of the great mysteries in modern medicine. Although none of the prevailing theories about the genesis of AD has resolved the mystery, each has led to intriguing findings suggesting the need for further investigations. AD is multifactorial, with both genetic and environmental factors implicated in its pathogenesis [4,22]. For a number of years two major hypotheses regarding the cause of AD were proposed. According to the amyloid cascade hypothesis the neurodegenerative process is a series of events triggered by abnormal processing of the amyloid precursor protein [1,14]. The neuronal cytoskeletal degeneration hypothesis assumes that cytoskeletal changes are the triggering events [32]. Much research on AD has focused on determining the mechanisms underlying the toxicity associated with Aβ proteins [25]. A major area of research into Aβ peptide toxicity is induction of inflammatory response following the release of pro-inflammatory cytokines that trigger an inflammatory cascade resulting in neuronal death [24]. Aβ peptide toxicity involves free radical formation. Synthesis of reactive oxygen species, which initiate cellular changes leading to apoptotic cell death, is regulated by multiple factors like calcium and inflammatory cascades [23,24]. Recent studies suggest an important role of blood vessels in the pathogenesis of AD. The microvascular aspects of neurodegenerative processes in AD were described, with special attention to cerebral blood flow, neuronal metabolic changes and the abnormalities of microvascular ultrastructure [8]. Numerous structural and functional alterations of the cerebral microvasculature in AD include increased microvascular density. More recently, a hypothesis regarding a crucial role of endothelial cells as mediators of progressive destruction of cortical neurons and pathologic angiogenesis in AD [33] caused much debate among investigators. This hypothesis points to neovascularization in the brain in AD that occurs in response to impaired cerebral perfusion and vascular injury. Morphological and biochemical evidence for this process include regionally increased capillary density, vascular loop formation, glomeruloid vascular structure formation and expression of angiogenic factors. The authors suggested that angiogenic activation of the brain endothelium in AD leads to deposition of β-amyloid plaques [33]. Recent findings implicate that perivascular neurons, astrocytes and vascular cells constitute a functional unit, acting together to maintain tight control of the biochemical composition of the brain parenchymal environment [16,38]. Multiple pathogenic cascades originating from altered cerebral blood vessels can initiate dysfunction of the neurovascular unit, including aberrant angiogenesis, cerebral amyloid angiopathy and senescence resulting in increased levels of Aβ [38]. Neovascularization during adult life has long been attributed to angiogenesis only. Studies on angiogenesis have revealed that adult bone marrow is a rich reservoir of endothelial progenitors. Mobilization and recruitment of these cells is essential for tissue revascularization in ischemic hindlimbs, ischemic myocardium, cutaneous wounds and tumor vasculature [2,21,30,31] in a process called postnatal vasculogenesis. In animal models of brain ischemia, immature endothelial cells were found to incorporate into sites of active neovascularization [2,18,27,35]. The aim of this study was to analyze the brain tissue from a patient with AD at the ultrastructural level using electron microscopy. We present evidence that vascular pathology in AD may be related to vascular progenitors.
Material
The patient, a 67-year-old woman, was urgently admitted to the Department of Neurosurgery in a very poor clinical status due to spontaneous intracerebral haematoma in the left cerebral hemisphere. Initially the patient was admitted to the Neurological Department in a regional hospital with suspected ischemic stroke. The patient had been treated for 4 years until that moment in a neurological outpatient clinic following the diagnosis of AD. CT of the head (Fig. 1) was performed due to worsening of the neurological status of the patient, leading to the diagnosis of intracranial haematoma and the patient was transferred to the Department of Neurosurgery. On admission her clinical condition was critical, scoring 7 points in GCS, with localizing response to pain only. A decision to perform emergent neurosurgical procedure was made. During the operation craniotomy was performed in the fronto-parietal area of the skull, and the haematoma was reached and removed through the cortex of the left cerebral parietal lobe. While making an access through the cerebral cortex, a small resection of the cerebral cortex was made due to surgical technique and the removed fragment was sent for microscopic examination. The clinical condition of the patient did not change after the surgery and she remained unconscious and intubated in a poor neurological status with hemiplegia. When the surgical wound healed, the patient was transferred to the regional Neurological Department. She remained in a critical condition, which unfortunately did not improve and after 4 weeks the patient died in the Neurological Department.
Methods
The material was processed for ultrastructural morphological studies using transmission electron microscopy and analyzed with JEM-1200EX as described earlier [9].
Results
The ultrastructural analysis of the fronto-temporal cortex specimens of our case revealed the presence of randomly oriented 7.5 to 9 nm amyloid fibrils in neurons and axons. In addition a large fibrillar deposit extending into the adjacent neuropil was observed (Fig. 2). Amyloid fibrils consisted of hollow rods and were composed of filaments arranged as a tightly coiled helix. At higher magnification they showed bead-like structure. The neurofibrillar tangles and dense bodies were observed in some parts of dystrophic neurites (Fig.3). The severe damage of the capillary walls has been observed in the fronto-temporal cortex of the affected patient. Microvascular changes were manifested by the pericytes and basement membrane abnormalities. Pericytes in our material showed signs of degeneration, including extensive deposition of lipids and granular dense material in phagolysosomes (Fig. 4). The basement membrane thickening probably due to the accumulation of basement membrane proteins and collagen deposition (Fig. 5) within basement membrane were the most frequent findings in investigated areas. The transverse diameter of the collagen fibrils was approximately 40 nm and the periodicity of fibrillar striation was 64 nm. The giant forms of collagen fibrils with a diameter about 100 nm, showing irregular profiles on transverse sections have been also observed in enlarged spaces between capillary vessels and the brain parenchyma. In some areas immature endothelial cells have been noted in small capillaries. They covered the surface of remaining endothelial cells (Fig. 6). In addition immature endothelial cells leaving the vessel lumen were observed in close proximity with the capillary wall. They were characterized by the presence of fine cytoskeletal fibrils and exhibited amyloid fibrils which decorated basement membrane-like material (Fig. 7). Cytoskeletal fibrils (a characteristic feature of the immature endothelial cell) and amyloid fibrils are morphologically different. Amyloid fibrils are seen as structureless material or poorly defined short rods or fuzzy fibrils (Fig. 7), whereas cytoskeletal fibrils form a regular dispersed network of intermediate filaments. Our observations show that the immature endothelial cells are rather added to the endothelium of preexisting blood capillaries than form new capillaries de novo. In many sections we failed to find endothelial progenitors beyond the limits of preexisting blood vessel wall. These cells did not leave the vessel and became stuck in the vessel wall, thus not creating any new vessels. Immature endothelial cells stuck in the vessels surrounded by thickened, blurred basement membrane formed a pattern of vascular loops with glomeruloid structure (Fig. 8).
Discussion
Characteristic pathological changes including thickening and accumulation of collagen fibrils in the basement membrane of capillaries in the brain were enhanced in AD as described earlier [7,17]. Horssen et al. [15] demonstrated that collagen type XVIII (non fibrillar collagen) is accumulated in amyloid-laden vessels and classic senile plaques and β-amyloid might trigger cells in the vessel wall to produce this specific type of collagen. Collagen type XVIII is capable of inhibiting endothelial proliferation and angiogenesis. Such ultrastructural alterations in brain capillaries are detected in various pathological conditions such as hypertension, diabetes, stroke, atherosclerosis, head injury, transient ischemia, or due to the effect of thrombogenic factors. All these conditions are associated with blood brain barrier discontinuity. The lack of correlation between the stage of the disease and the aberrations of cerebral capillaries suggests that the changes in microvasculature are not a consequence of AD pathology [19]. One may wonder, however, whether AD results from vascular damage or dysfunction. Endothelial damage, pathological evidence of disruption of blood brain barrier and β-amyloid deposition in brain vessels are almost universal in the advanced stages of AD. The blood vessel theory has been expanded to hypothesize that potential defects in the blood-brain barrier are a result of serious head injuries. Although neuronal cell death is the pathologic evidence of AD, it is difficult to clarify the primary mechanism resulting in this pathology. The hypothesis that cerebral microvasculature is a key factor in the pathogenesis of AD has been suggested earlier [12,26,29]. Although there is no evidence showing functional impairment of blood brain barrier permeability or transient alteration of BBB integrity during aging, resistance to endogenous albumin transvasation by microvascular systems adjacent to amyloid deposits [36] and BBB leakage was observed in AD [13]. The vascular pathology has been described in AD but relatively little is known about the pathogenic mechanisms by which brain endothelial cells contribute to dementia and lesions in AD brains. According to the current concepts, the brain vascular system is continually modified in order to maintain adequate cerebral blood flow and brain perfusion, so normal laminar flow in brain capillary vessels becomes disturbed in regions where discontinuity of BBB and ultrastructural changes in capillaries occur [5,6]. What factors underlie disturbed blood flow in AD? The most interesting feature of analyzed material was new vessel formation. According to current concepts regarding new vessel formation, circulating cells are present in the peripheral blood that may contribute to neoangiogenesis in adults consistent with vasculogenesis. The ultrastructural features of these cells were described earlier by us in a surgical model of brain trauma [9] and confirmed by Flk-1 expression [10]. We can thus suggest that the pathological aspect of angiogenesis in AD is related to the perpetuating process of remodeling in most blood vessels, mediated by vascular progenitor cells and leading to decrease in the vessel lumen. Our ultrastructural findings are confirmed by earlier studies [20] that pointed to the absence of specific endothelial cell markers (CD34 and CD31) in AD-related degeneration. The endothelial damage may be a functional disturbance rather than a result of cellular attachment and it seems that the process will continue as long as the injury is present. There is also a possibility that inflammatory factors that are elevated in AD [11] and destroy capillary walls are linked to a deficient repair process mediated by cytokines released by premature endothelial cells. Endothelial cytotoxicity induced by cytokines will increase blood brain barrier permeability [34]. The endothelial progenitor attachment to adult endothelium may result in blood vessel injury and induce formation of cytokines and other molecules. These molecules trigger not only a paracrine effect on the adjacent vessel wall but also an endocrine signal that is received by responsive bone marrow cells [28]. It was observed that with aging, and in the presence of risk factors, the progenitors originating from the marrow become incompetent, resulting in a loss of their capacity for repair of the vessel wall, which is in turn becomes dysfunctional. This was confirmed by our ultrastructural findings. If new vessel formation is an adaptative process in many pathologic situations associated with BBB discontinuity or hypoperfusion, endothelial precursors circulating in the blood and participating in that adaptation become an important element in AD. The mechanisms of repair and associated vascular growth are still poorly understood in the context of adaptation processes leading to neurodegenerative disorders. As was shown in our studies, immature endothelial cells contain amyloid fibrils in cytoplasm. Some researchers discussed specific aspects of vascular dysfunction in AD related to aberrant transport of Aβ across the BBB and its role in the development of cerebral amyloidosis [37]. In these assumptions the BBB transport dysfunction for Ab is a late event in AD, possibly resulting from disease-specific vascular disorder of brain endothelium associated with aberrant angiogenesis, cellular senescence and altered expression of a subset of genes. Our observations indicate that immature endothelial cells may be an important source of circulation-derived amyloid in the brain. The consequence of immature endothelial cells should be appreciated more fully in both a pathobiological and therapeutic sense. While data regarding putative therapeutic potential of endothelial progenitors continue to mount, it is perhaps not improper to ask what is the biological context and time frame of their mobilization and activity. In this sense, features and impact of endothelial progenitors will probably change with better understanding of genetic, phenotypic and microenvironmental characteristics of a given disease. The angiogenesis was initially only implicated in cancer, arthritis and psoriasis, but recently it has become evident that excessive, insufficient or abnormal angiogenesis contributes to the pathogenesis of many more disorders. The same may apply to immature endothelial cells.
References
1. Armstrong RA. Is there a spatial association between senile plaques and neurofibrillary tangles in Alzheimer’s disease? Folia Neuropathol 2005; 43: 133-138. 2. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964-967. 3. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearney M, Magner M, Isner JM. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221-228. 4. Barcikowska M. Terapeutic approaches in Alzheimer’s disease. Folia Neuropathol 2004; 42: 251-255. 5. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nature Med 2000; 6: 389-395. 6. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 8: 242-248. 7. Farkas E, De Jong GY, de Vos RA, Jansen Steur EN, Luiten PG. Pathological features of cerebral cortical capillaries are doubled in Alzheimer’s disease and Parkinson’s disease. Acta Neuropathol 2000; 100: 395-402. 8. Farkas E, Luiten PG. Cerebral microvascular pathology in aging and Alzheimer’s disease. Progress in Neurobiology 2001; 64: 575-611. 9. Frontczak-Baniewicz M, Walski M. New vessel formation after surgical brain injury in rat’s cerebral cortex. Formation of the blood vessels proximally to the surgical injury. Acta Neurobiol Exp (Wars.) 2003; 63: 65-75. 10. Frontczak-Baniewicz M, Gordon-Krajcer W, Walski M. The premature endothelial cell in new vessel formation following surgical injury in rat brain. Neuroendocrinology Letters, in press. 11. Grammas P, Ovase R. Inflammatory factors are elevated in brain microvessels in Alzheimer’s disease. Neurobiol Aging 2001; 22: 837-842. 12. Grammas P, Yamada M, Zlokovic B. The cerebromicrovasculature: A key player in the pathogenesis of Alzheimer’s disease. J Alzheimer’s Dis 2002; 4: 217-223. 13. Hachinski V, Munoz DG. Cerebrovascular pathology in Alzheimer’s disease: cause, effect or epiphenomenon? Ann NY Acad Sci 1997; 826: 1-6. 14. Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade hypothesis. Review Science 1992; 256: 184-185. 15. Horssen van J, Wilhelmus MMM, Heljasvaara R, Pihlajaniemi T, Wesseling P, Waal de RMW, Verbeek MM. Collagen XVIII: a novel heparin sulfate proteoglycan associated with vasculat amyloid depositions and senile plaques in Alzheimer’s disease brains. Brain Pathol 2002; 12: 456-462. 16. Iadecola C. Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat Rev 2004; 5: 347-360. 17. Inoue S, Kuroiwa M, Kisilevsky R. Basement membranes, microfibrils and b amyloid fibrillogenesis in Alzheimer’s disease: high resolution ultrastructural findings. Brain Res Rev 1999; 29: 218-231. 18. Isner JM. Angiogenesis: a “breakthrough” technology in cardiovascular medicine. J Invasive Cardiol 2000; Suppl A: 7A-14A. 19. De Jong GI, De Vos RA, Steur EN, Luiten PG. Cerebrovascular hypoperfusion: a risk factor for Alzheimer’s disease? Animal model and postmortem human studies. Ann NY Acad Sci 1997; 826: 56-74. 20. Kalaria RN, Hedera P. Differential degeneration of the cerebral microvasculature in Alzheimer’s disease. Neuroreport 1995; 6: 65-74. 21. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 2000; 97: 3422-3427. 22. Kowalska A. Genetic aspects of amyloid beta-protein fibrillogenesis in Alzheimer’s disease. Folia Neuropathol. 2004; 42: 235-237. 23. Law A, Gauthier S, Quirion R. Say NO to Alzheimer’s disease: the putative links between nitric oxide and dementia of the Alzheimer’s type. Review. Brain Res Rev 2001; 35: 73-96. 24. Leonard BE. Changes in the immune system in depression and dementia: causal or co-incidental effects? Int J Dev Neurosci 2001; 19: 305-312. 25. Pearson HA. Alzheimer’s Disease. In: Hopper NM (ed.). Methods and Protocols, Humana Press, Totowa, NJ, 2000; pp. 113-138. 26. Perlmutter LS, Chui HC. Microangiopathy, the vascular basement membrane and Alzheimer’s disease: A review. Brain Res Bull 1990; 24: 677-686. 27. Rafii S. Circulating endothelial precursors: mystery, reality, and promise. J Clin Invest 2000; 105: 17-19. 28. Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, Wang T, Gregg D, Ramaswami P, Pippen AM, Annex BH, Dong C, Taylor DA. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003; 108: 457-463. 29. Rhodin JA, Thomas T. A vascular connection to Alzheimer’s disease. Microcirculation 2001; 8: 207-220. 30. Schatteman GC, Hanlon HD, Jiao C, Dodds SG, Christy BA. Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice. J Clin Invest 2000; 106: 571-578. 31. Takahashi T, Kalka C, Masuda H Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999; 5: 434-438. 32. Terry RD. The cytoskeleton in Alzheimer disease. Review. J Neural Transm Suppl. 1998; 53: 141-145. 33. Vagnucci AH Jr, Li WW. Alzheimer’s disease and angiogenesis. The Lancet 2003; 361: 605-608. 34. de Vries HE, Blom-Roosemalen MC, van Oosten M, de Boer AG, van Berkel TJ, Breimer DD, Kuiper J. The influence of cytokines on the intergrity of the blood-brain barrier in vitro. J Neuroimmunol 1996; 64: 37-42. 35. Weinstein BM. What guides early embryonic blood vessel formation? Dev Dyn 1999; 215: 2-11. 36. Wisniewski HM, Vorbrodt AW, Wegiel J. Amyloid angiopathy and blood-brain barrier changes in Alzheimer’s disease. Ann NY Acad Sci 1997; 826: 161-172. 37. Zlokovic BV. Vascular disorder in Alzheimer’s disease: role in pathogenesis of dementia and therapeutic targets. Advanced Drug Delivery Rev 2002; 54: 1553-1559. 38. Zlokovic BV. Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosc 2005; 28: 202-208.