Evidence of Cardiac Disease in HIV-1 Infected Humanized Mice

Nathan Zaroban, Isabella Catalano, Prasanta Dash, PhD, Santhi Gorantla, PhD
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical
Center, Omaha, NE 68198
Introduction

Materials and Methods

A.

People infected with HIV-1 are at a greater risk of cardiovascular disease (CVD)
than non- infected people. 3,6,8

Immunohistology. Five micron thick longitudinal sections were cut from paraffin
embedded heart tissues. Immunohistochemical staining was performed on the
sections using HLA-DR, CD68, HIV-1 p24 gag protein, and -SMA antibodies. HLADR stains for all human immune cells, CD68 stains for human macrophages, and
p24 stains for HIV-1 infected cells. -SMA stains for smooth muscle actin. The
secondary antibodies conjugated to HRP were used and the sections were
developed using DAB. Antibodies came from Dako, Carpinteria, CA.

Title

Other risk factors associated with HIV-1 infected persons such as long-term
antiretroviral drug treatments (ART), opportunistic infections, recreational drug
abuse, and increasing age, all may increase the risk of CVD. 4 Because of these
multiple factors, it is difficult to study HIV-1 associated CVD in humans.
HIV-1 specifically infects human immune cells, thus precludes utilization of
standard animal models to study HIV-1 pathogenesis. Humanized mice
reconstituted with a functional human immune system are susceptible to HIV-1
infection, and chronically infected mice mirror human pathology.5

Cardiomyopathy is one of the most common CVDs found in HIV-1-infected

individuals. 7 Cardiomyopathy leads to a deterioration of the hearts ability to
contract, which increases the risk of heart attack and stroke.

Alpha smooth muscle actin (-SMA) is a protein marker for myofibroblast cell
activation.1,2 The role of myofibroblast cells is to collagenate damaged muscle
tissue areas. Upregulation of -SMA has been found to positively correlate with
the degree of collagenation.1,2

Evidence of Cardiac Fibrosis

We hypothesized that humanized mice infected with HIV-1 would be a suitable

model to study HIV induced cardiovascular abnormalities related both to HIV alone
and additional factors such as anti-retrovirals, co-infections and drugs of abuse.

Modified Massons Trichrome Staining. Five micron thick longitudinal sections

were stained according to the protocol provided by the ScyTek Laboratories, Inc.
Modified Massons Trichrome Stain Kit. Trichrome staining differentiates between
smooth muscle tissue and fibrous collagen.
Imaging and Optical Density Quantification. Images were captured using a Nikon
ECLIPSE 55i microscope and a CRI Nuance Multispectral Imaging System. Optical
density quantification was performed using CRI Nuance Multispectral Imaging
software.

HIV-1 Infection in Humanized Mice

Mouse
Number

Time of Infection
(Weeks)

Viral RNA
Copies/mL

2725

1.35 x 10^5

2726

3.91 x 10^5

2727

1.25 x 10^6

2744

1.24 x 10^6

2748

1.09 x 10^6

2749

9.35 x 10^5

2753

6.8 x 10^3

2025

13

2.6 x 10^5

1955

13

3.9 x 10^4

1957

13

1.15 x 10^5

H16

13

3.47 x 10^5

Table 1. Post-sacrifice HIV-1 viral load in

blood of infected humanized mice.

HIV-1 Infection. Humanized mice, at 20 weeks of age, were infected with HIV-1ADA by
intraperitoneal injection of 105 tissue culture infectious dose50 (TCID50). Mice were sacrificed
at 4, 6, and 13 weeks after infection. Blood was collected to analyze human immune cell
levels using flow cytometry. HIV-1 viral loads were detected in plasma using a Roche
COBAS Amplicor. Organs were harvested, fixed in 4% paraformaldehyde, and processed for
paraffin embedding.

Figure 3. CD4+ T lymphocyte depletion in HIV-1 infected mice

measured using flow cytometry at 20 weeks of age (pre-infection)
and at the time of sacrifice. Error bars= SD. CD4+ T lymphocyte
depletion is characteristic to HIV-1 infection because the virus
specifically binds to the CD4 receptor. *p<.0001

Infected

Uninfected

-SMA

Trichrome
Collagen
Staining

C.

Figure 4.
A. Infiltration of HLA-DR+ human immune
cells in heart section. Error bars=SD.
*p=.0293
B. HLA-DR+ (brown) cells in heart sections
(40x).
C. CD68+ (brown) macrophages in heart
sections (40x).
D. p24+ (brown) cell in heart section (40x)

Figure 5. A. Heart sections from uninfected and HIV infected mice were stained for a-SMA (brown)
by immunohistology and Massons Trichrome staining for collagen (Blue = fibrous collagen, red =
myocytes, black = nuclei). Magnification 40x. B. Intensity of a-SMA was quantified by CRI Nuance
Multispectral Imaging System. Statistical significance by one-way anova. Error bars= SD.
C. Correlation analyses between a-SMA expression and number of heart infiltrated immune cells or
peripheral viral load.

Conclusions and Future Directions

Increased immune cell infiltration into the heart muscle and presence of
macrophages in the myocardial fibers of HIV-1 infected mice might be
due to HIV-1 induced inflammation. Further characterization of cytokine
expression is required.
Occurrence of mild cardiac fibrosis might be due to HIV-1 infection.
Reactive myofibroblasts expressing a-SMA are found during acute HIV
infection and perivascular fibrosis is observed with chronic infection.
Functional assays on live infected mice to detect cardiac muscle
dysfunction will help to understand the disease progression.

References and Acknowledgements

Figure 2. Timeline of mouse humanization, HIV-1 infection, and sacrifice.

B.

HIV-1 infection in humanized mice mirrors human pathogenesis with

sustained viral loads and CD4+ T lymphocyte depletion.

Infiltration of Human Immune Cells in Heart Tissue

A

Figure 1. Overview of mouse humanization process.

Infected

Results

Materials and Methods

Humanized Mice. NOD/Scid/IL2-/- (NSG) mice
were irradiated with a sub-lethal dose (1Gy) at birth
and intrahepatically injected with 104 CD34+
hematopoietic stem cells (HSCs). The HSCs were
isolated from human umbilical cord blood using
Miltenyi magnetic bead method. At 10 weeks of age,
mice were bled and tested for successful human
reconstitution using flow cytometry (BD LSR II).
Antibodies for human CD45, CD3, CD4, CD8, CD19,
and CD14 cells were used to measure human
leukocyte, T and B lymphocyte, monocytes and
macrophage. Antibodies were purchased from BD
Biosciences.

Uninfected

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208.
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I would like to thank Dr. Santhi Gorantla and Dr. Larisa Poluektova for their guidance and giving me the opportunity to work in
their lab. I would also like to thank lab members Edward Makarov, Raghubendra Dagur, Phd, Weizhe Li, Li Wu, Yan Cheng,
Amanda Branch Woods, Aditya Bade, Divya Prakash, PhD, Weimin Wang, MD, and Amy Conaway for their continued guidance,
assistance and patience. Finally, I would like to thank the UNMC PEN department and Dr. Gendelman for the opportunity to
participate in the SURP program.