Kamal Narayana

Topic

Serpine1/PAI-1 role on blood flow, stalling and vessel width in stroke risk and recovery

Division of Medical Sciences

Date & location

• Friday, August 16, 2024
• 10:00 A.M.
• Medical Sciences Building, Room 150

Examining Committee

Supervisory Committee

• Dr. Craig Brown, Division of Medical Sciences, University of Victoria (Supervisor)
• Dr. Andy Shih, Center for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute (Outside Member)

External Examiner

• Dr. Gergely Silasi, Department of Cellular and Molecular Medicine, University of Ottawa

Chair of Oral Examination

• Dr. Michael Raven, Department of Philosophy, UVic

Abstract

The brain relies heavily on the proper function of microvascular hemodynamics such as blood flow for sufficient oxygenation and clearance of metabolic waste. Therefore, it is no surprise that damage to blood vessels can result in heterogenous blood flow and obstructions furthering the effects of injury. Ischemic stroke can lead to a long-lasting disruption of blood flow in microvessels surrounding the infarct site, exacerbating injury beyond the initial insult. Homeostatic blood clotting and proteolytic (clot-busting) pathways are likely fundamental to regulating post-ischemic capillary blood flow, and thus functional recovery. We have recently discovered that the SERPINE1 (Serp1) gene, encoding for Plasminogen Activator-Inhibitor-1 (PAI-1), is highly expressed along blood vessels following photothrombotic stroke in the rodent somatosensory cortex. Therefore, we explored the role of SERPINE1/PAI-1 on cortical blood flow and its potential downstream effects following experimentally induced ischemic stroke.

In the first aim, we examined the spatial and temporal expression of Serp1 post-stroke across multiple days using immunohistochemistry and confirmed with whole tissue RNA sequencing. We discovered that Serp1/PAI-1 expression is highly upregulated 3-days post-stroke (i.e., subacute), and interestingly, this expression was brain-wide. We also obtained RNA levels at 3-days confirming an upregulation in Serp1 post-stroke. We then successfully performed an endothelial-specific knockdown (KD) of Serp1 which we confirmed using RNA seq. In the second aim, using in vivo 2-photon imaging, we longitudinally imaged superficial cortical blood flow in adjacent and distant areas to the infarct in both Serp1+/+ and cerebral endothelial Serp1 KD mice (Serp1-/-). We discovered reduced blood flow in the Serp1-/- in the penumbra and distant regions following stroke in the subacute (3d) and chronic phase (35d). The effects of the reduced blood were prominent in arteriole capillaries, and surprisingly, blood flow did not recover in Serp1-/-. In the third aim, we measured vessel width in the penumbra and distant regions across the imaging days and separated them by arteriole and venule capillaries. Surprisingly, we found that arteriole and venule capillaries in Serp1-/- were constricted in the subacute and chronic phase post-stroke, whereas in Serp1+/+, capillaries were dilated in the subacute phase only. These respective effects mimicked the observed heterogenous blood flow (increase in Serp1+/+ and decrease in Serp1-/-) between the genotypes. In the fourth aim, we used in vivo 2-photon imaging to longitudinally label leukocytes with anti-CD45.2 to distinguish between the type of stalling. Surprisingly, we found that, alongside Serp1+/+, the total number of stalls increased in Serp1-/- mice, despite the presumed coagulant role of PAI-1, which was mediated by a greater number of leukocyte stalls over red-blood cell (RBC) stalls. In the fifth aim, we analyzed neuroinflammatory gene expression in both Serp1+/+ and Serp1-/- mice in the subacute phase post-stroke. We found an asymmetrical distribution in favor of upregulating neuroinflammatory genes as an effect of stroke with Serp1 as one of the top genes in Serp1+/+. Interestingly, the effect of Serp1 KD overall reduced the fold change of the genes highly expressed in Serp1+/+ indicating for a potential beneficial and protective role of the KD.

These findings reveal that Serp1 KD significantly affects microvascular hemodynamics and leukocyte recruitment, suggesting that merely increasing clot degradation (i.e., tissue plasminogen activator (tPA) treatment) following stroke may not necessarily improve local blood flow to penumbra. In combination with the genomic changes observed, Serp1 KD shifted the distribution of the neuroinflammatory genes, which may also suggest for a protective role in KD post-stroke. Altogether, Serp1 KD elucidated a complex set of microvascular-related and genomic changes that requires further investigation into its role in stroke recovery.