**4. Concluding remarks**

In this chapter, we have outlined many lines of evidence linking the activity of astrocytes to changes in the ECM associated with neuropathology. We postulate that establishing the nature of astrocyte heterogeneity will be important for understanding the growing number of diseases in which astrocytes have been identified as having a primary or causal role. The growing awareness that astrocyte dysfunction, not just reactivity, contributes to neuropathology as a concept we have called "gliodystrophy" [152]. This term reflects more than the presence of astrocytes in pathology, but denotes the loss or gain of astrocyte functions as a result of astrocyte plasticity and disease-associated heterogeneity. Therefore, understanding the basis for astrocyte heterogeneity, as a component of astrocyte dysfunction, is of increasing impor‐ tance as astrocytes are recognized to play a central role in a wide range of neurological diseases.

Laminin also plays a role in maintaining the integrity of the blood-brain barrier (BBB). The BBB is a dynamic network that regulates material exchange between the circulatory system and the brain parenchyma, which aids in homeostatic maintenance of the central nervous system [147]. In the context of central nervous system injury, BBB malfunction has been reported. The BBB is mainly composed of brain microvascular endothelial cells, astrocytic endfeet, pericytes, and the basement membrane, of which laminin is a key component. Astrocytes wrap around endothelial cells using their end-feet, and pericytes, which are sandwiched between endothelial cells and astrocytes, signal to both cell types. Recently, it has been shown that pericytes are necessary for the formation of the BBB during embryogenesis, and loss of pericytes leads to comprised BBB integrity and age-dependent vascular-mediated neurode‐ generation in adult mice, which suggests an important role for pericytes in BBB regulation. In a recent report, a group found that astrocyte laminin, by binding to the integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, which helps to maintain the integrity of the BBB [148]. However, when astrocytic laminin was down-regulated using functional blocking antibodies and RNA interference, there were decreases in aquaporin-4 expression on astrocyte end-feet and decreases in tight junction protein expression. Further, in laminin knockdown animals, the lack of astrocytic laminin induced the differentiation of pericytes from the resting stage to the contractile stage. This loss of astrocytic laminins could be one of the major driving forces behind the leakiness of the BBB seen in many neurodegenerative diseases and CNS injuries.

Unlike the preceding extracellular matrix proteins, vitronectin has remained elusive in its functional role in central nervous system inflammation and injury. The earliest reports observed an enhancement of vitronectin expression in the blood vessel walls of active demye‐ linating lesions, in demyelinated axons, and on a small number of hypertrophic astrocytes. However, a negative role for vitronectin has not been found. In contrast, vitronectin has been shown to promote neurite outgrowth [149] and enhance astrocyte migration [150]. As vitro‐ nectin mRNA is almost undetectable in the normal adult brain, it might be synthesized by infiltrating leukocytes or derived from the plasma as a result of blood-brain barrier breakdown. In the EAE model of multiple sclerosis, vitronectin expression was shown to be enhanced, as well as contribute to the up-regulation of matrix metalloproteinases and activation of microglia [151]. Increasing research into the role of this under-studied extracellular matrix protein could

In this chapter, we have outlined many lines of evidence linking the activity of astrocytes to changes in the ECM associated with neuropathology. We postulate that establishing the nature of astrocyte heterogeneity will be important for understanding the growing number of diseases in which astrocytes have been identified as having a primary or causal role. The growing awareness that astrocyte dysfunction, not just reactivity, contributes to neuropathology as a

**3.4. Vitronectin in CNS disease and injury**

126 Composition and Function of the Extracellular Matrix in the Human Body

**4. Concluding remarks**

provide clues as to its functional role in CNS inflammation.

How might we define astrocyte heterogeneity and is astrocyte reactivity a form of heteroge‐ neity? To begin, we would propose that the heterogeneity of astrocytes is divergence in functions between ontogenically identical cells. By this definition, we would suggest that there would exist homeostatic heterogeneity among astrocytes related to their anatomical location. One could argue that the metabolic and physiological demands on an astrocyte within the cortical gray matter would be different from an astrocyte located within the heavily myelinated tracts of the corpus callosum. Indeed, astrocytes in these two locations are easily discernible by their overt appearance as either protoplasmic or fibrous [92]. Then, reactivity would add another dimension to this heterogeneity as each would, in theory, have potentially unique starting states from which reactivity could also be distinctive. If we depict this idea in a (perhaps overly) simplified manner (**Figure 2**), we could envision naive astrocytes within the CNS lying along an X-axis at different points. In response to a stimulus, you might then predict that each cell would in response to that trigger rise up the Y-axis to different points. When considered in terms of neurological diseases, identical acute injuries or trauma to different anatomical loci may be expected to evoke different responses from astrocytes in terms of their proliferation, gene response, and secretome contribution to the immediate environment. When considered in terms of chronic neurodegenerative conditions, where time plays a crucial role (as conveyed along the Z-axis in **Figure 2**), heterogeneous astrocytes may be expected to develop adaptations to disease in different ways. Where one activated cell may become quiescent, interacting less with its immediate environment and failing to sustain homeostatic functions, as one might envision occurring in a glial scar. Another astrocyte may instead adopt a gain of function with an enhanced or altered secretome that modifies its immediate envi‐ ronment and interacts with adjacent cells in a pathological way. This concept may contribute to how we might explain the role of astrocytes in dementia where dysregulation of synaptic connectivity and impaired cognitive function may relate to astrocyte senescence in Alzheim‐ er's disease [153].

From all of this discussion, we should also consider the potential utility of the information gleaned from what could be considered the basic biology of the astrocyte. How could we apply our present and future understanding of astrocyte heterogeneity to developing new, or possibly enhancing current treatments for neurological disease? One possible approach of harnessing the potential of heterogenous astrocytes has already been applied to models of spinal cord injury and Parkinson's disease. In these studies, Proschel and colleagues have determined that astrocyte transplants can dramatically improve the outcomes in these degenerative conditions. For instance, in 6-hydroxydopamine lesioned rats, the behavioral deficit and dopaminergic denervation of the striatum were attenuated when these animals received transplants of astrocytes pre-exposed to bone morphogeneic protein [154]. In a previous study, this group also demonstrated enhanced axon growth in a spinal cord injury model with these astrocyte transplants [155]. These data show that astrocytes possess thera‐ peutic potential to address important neurological diseases. To build upon the ideas set forth by these transplant studies, one could ask how could we target the endogenous astrocytes to achieve similar outcomes? While the answer to this important question is likely complex, if our own ideas on the origins of astrocyte heterogeneity are valid, then select targeting of ECM-Astrocyte communication may be one approach to try. For instance, targeting of the β1 integrin using the RGD peptide has been shown to prevent astrogliosis in the injured spinal cord and improve functional recovery [156]. With an advanced understanding on how the ECM controls, or at the very least influences, the function of astrocytes *in situ* during brain injury or disease, we may be able to target and promote brain recovery and repair.

In the future, we suggest that technical approaches are now available to advance this line of investigation in ways not previously feasible. For instance, cataloging astrocyte diversity using single-cell laser-capture sequencing may be expected to identify unique markers to distinguish different subtypes of astrocytes from tissues. This approach would also allow for the important distinction of acquiring astrocytes that are spatially and temporally associated with specific types of neural injury [157]. A similar approach has recently been used to identify markers of reactive astrocytes. Results from these types of future investigations should enable us to delve deeper into the complexity of astrocyte biology and better understand the nature and function of these cells as they maintain the CNS and react and participate in neurological disease states.

**Figure 2.** Hypothesized model of influence of ECM on innate and acquired astrocyte heterogeneity. Depicted are two different astrocytes, labeled A and B which have been positioned along the X-axis to reflect innate heterogeneity on the basis of their location within the central nervous system (CNS). In response to a stimulus (labeled A' and B', respec‐ tively), the innate heterogeneity impacts the reactivity, as depicted as different locations along the Y-axis. Lastly, with chronic stimulation, these two distinct cells develop distinct long-term phenotypes, labeled A" and B", where the in‐ nate heterogeneity results in different outcomes to the long-term stimulation. Whereas A depicted as a smaller sphere lower on the Y axis may become chronically less active, perhaps related to development of an astrocyte scar, the other astrocyte labeled B" with chronic stimulation adapts to become a more active, perhaps physiologically adapted, phe‐ notype contributing to neuropathology in disease.
