Therapeutic Remodeling of Postnatal Cerebrovasculature

Arinzechukwu Nkemdirim Okere

Mentor: Dr. Grant Anderson, PhD

University of MinnesotaCollege of Pharmacy, Duluth

Mom and Daughter Doing Great

Older Sister,not too sure

The human cerebrovasculature

Neurosurgery 43:877, 1998 Zlokovic and Apuzzo

Microvessels of the Human Cerebral Cortex

Akima et al, Lab. Invest. 55:482 (1986)

(courtesy of Lester Drewes)

Electron micrograph of a brain capillary in cross-section

Pathophysiology and the Blood Brain Barrier (BBB)

• BBB and CNS drug delivery• Stroke and brain tumors • Genetic disorders of the brain endothelial cell

Glut-1 Deficiency• Glut-1 mediates the transport of glucose across the

luminal brain endothelial cell outer membrane• Glut-1 is the dominant glucose transporter expressed in

the brain endothelial cell• Functional mutations of the Glut-1 gene lead to impaired

glucose transport to the brain (Glut-1 deficiency syndrome)

• Glut-1 deficiency syndrome was first described in 1991 as a sporadic clinical condition– Haploinsufficiency– Clinical characteristics include

• Infantile seizures,developmental delay, acquired microcephaly, spasticity, ataxia, and hypoglycorrhachia

– No known treatment for this orphan disease

Clinical Goals

• To correct Glut 1 deficiency by populating the brain cerebrovasculature with wild type Glut-1 expressing endothelial cells

• Use the developed methodology to provide a therapeutic approach for treating other brain disorders

Definitions

• Neovasculogenesis: Outgrowth of new blood vessels– Angiogenesis: Outgrowth of new blood vessel via

preexisting endothelial cell (EC) proliferation– Vasculogenesis: Outgrowth of new blood vessel via

endothelial progenitor cell (EPC) incorporation and proliferation

Hypotheses

• Physiologic response of the brain cerebrovasculature to chronic mild hypoxia– Neovasculogenesis

• Hypotheses– Hypoxia induced brain neovasculogenesis is resultant

from the process of vasculogenesis– Isolated EPCs i.v. infused during chronic mild hypoxia

treatment will home to the brain and form nascent blood vessels

– Genetically engineered EPCs infused during chronic hypoxia will allow therapeutic engineering of the BBB

Overview of Research Project

Incorporate Glut-1 ACE enginneered ex vivo cultured EPCs into brain neovasculature

Incorporate ACE engineered ex vivo cultured EPCs into brain neovasclature

Assess contribution of vasculogenesis to hypoxia induced brain neovasculature

Establish hypoxia induced brain neovasculogenesismethod

Incorporate ex vivo cultured EPCs into brain neovasculature

My Project Goals

Develop brain vascular morphometric assay

Create a hypoxic hypoxia environment chamber at 10% normobaric oxygen

Use morphometric assay to validate our chronic hypoxia system

Approach1. Construct normobaric hypoxic hypoxia chamber 2. Develop brain morphometric assay

a) Establish Glut-1 immunohistochemical staining method• Fix isolated brains in 4% paraformaldehyde• Paraffin embed tissues and slice 8μm sections with a

microtome• Optimize conditions for Glut-1 immunohistochemistry• Develop morphometric method on Glut-1 stained brain tissue

sections3. Use morphometric assay to validate our chronic hypoxia

systema) Mice were subjected to chronic hypoxia of 10% Oxygen for 3

weeks in a normobaric hypoxic chamber.b) Tissues harvested, fixed and subjected to Glut-1

immunohistochemistryc) Assess physiologic markers of chronic hypoxiad) Conduct morphometric analysis on brain microvessels

Approach1. Construct normobaric hypoxic hypoxia chamber 2. Develop brain morphometric assay

a) Establish Glut-1 immunohistochemical staining method• Fix isolated brains in 4% paraformaldehyde• Paraffin embed tissues and slice 8μm sections with a

microtome• Optimize conditions for Glut-1 immunohistochemistry• Develop morphometric method on Glut-1 stained brain tissue

sections3. Use morphometric assay to validate our chronic hypoxia

systema) Mice were subjected to chronic hypoxia of 10% Oxygen for 3

weeks in a normobaric hypoxic chamber.b) Tissues harvested, fixed and subjected to Glut-1

immunohistochemistryc) Assess physiologic markers of chronic hypoxiad) Conduct morphometric analysis on brain microvessels

Mount Everest South Col base camp 16,000 feet

Mount Everest Base Camp

The city of Puno on the Peruvian Altiplano plain (12,420 feet)

Potala Palace, Lhasa Tibet (12,100 feet)

Horsemen on the Tibetan plains

Hypoxic Hypoxia Chamber (elevation outside chamber = 607 feet (20.5 % oxygen), relative elevation inside chamber =15,000 feet (10% oxygen)

Everest Summit

Mouse mountaineers

Sherpa

Approach1. Construct normobaric hypoxic hypoxia chamber2. Develop brain morphometric assay

a) Establish Glut-1 immunohistochemical staining method• Fix isolated brains in 4% paraformaldehyde• Paraffin embed tissues and slice 8μm sections with a

microtome• Optimize conditions for Glut-1 immunohistochemistry• Develop morphometric method on Glut-1 stained brain tissue

sections3. Use morphometric assay to validate our chronic hypoxia

systema) Mice were subjected to chronic hypoxia of 10% Oxygen for 3

weeks in a normobaric hypoxic chamber.b) Tissues harvested, fixed and subjected to Glut-1

immunohistochemistryc) Assess physiologic markers of chronic hypoxiad) Conduct morphometric analysis on brain microvessels

Glut-1 immunofluorescence identifies brain microvessels

Glut-1 positive microvessels

Nuclei

Morphometric method

Approach1. Construct normobaric hypoxic hypoxia chamber 2. Develop brain morphometric assay

a) Establish Glut-1 immunohistochemical staining method• Fix isolated brains in 4% paraformaldehyde• Paraffin embed tissues and slice 8μm sections with a

microtome• Optimize conditions for Glut-1 immunohistochemistry• Develop morphometric method on Glut-1 stained brain tissue

sections3. Use morphometric assay to validate our chronic hypoxia

systema) Mice were subjected to chronic hypoxia of 10% Oxygen for 3

weeks in a normobaric hypoxic chamber.b) Tissues harvested and fixed c) Assess physiologic markers of chronic hypoxiad) Conduct morphometric analysis on brain microvessels

Hematocrit, hemoglobin and ceruloplasmin levels are all elevated after 3 weeks of mild hypoxia

***

p<0.0002

*

p<0.027

p<0.0074

**

HypoxicNormoxic

**

p<0.0019

Morphometric analysis reveals cortical neovasculogenesis

p<0.042

Granular Layer

HypoxicNormoxic

Morphometric analysis reveals cerebellarneovasculogenesis

*

p<0.42

Molecular Layer

Overview of Research Project

Incorporate Glut-1 ACE enginneered ex vivo cultured EPCs into brain neovasculature

Incorporate ACE engineered ex vivo cultured EPCs into brain neovasclature

Assess contribution of vasculogenesis to hypoxia induced brain neovasculature

Establish hypoxia induced brain neovasculogenesismethod

Incorporate ex vivo cultured EPCs into brain neovasculature

Acknowledgements

• Dr.Grant Anderson– Dan Westholm– Kevin Viken

• Members of Hypoxia Group– Dr. Les Drewes– Dr. Ed Perkins

• Funding support for this project– Arinze Okere

• Office of Clinical Research AHC-University of Minnesota

– Grant Anderson• 2007 AHC Faculty Seed Grant, NIH R21